U.S. patent application number 17/296379 was filed with the patent office on 2022-02-03 for nanocarrier systems for imaging and delivery of active agents.
The applicant listed for this patent is Kansas State University Research Foundation. Invention is credited to Santosh Aryal, Tuyen DT Nguyen, Arunkumar Pitchaimani.
Application Number | 20220031869 17/296379 |
Document ID | / |
Family ID | 70853653 |
Filed Date | 2022-02-03 |
United States Patent
Application |
20220031869 |
Kind Code |
A1 |
Aryal; Santosh ; et
al. |
February 3, 2022 |
NANOCARRIER SYSTEMS FOR IMAGING AND DELIVERY OF ACTIVE AGENTS
Abstract
Synthetic nanocarrier constructs and related compositions
comprising a lipid-based bilayer membrane infused with one or more
NK-92 cell membrane proteins, which encapsulates a liquid receiving
interior space or coats at least a portion of a solid core. Methods
of targeted delivery of an active/diagnostic/imaging agent to a
specific cell type or a region of a patient by administering a
plurality of nanocarrier constructs to the patient. MRI imaging
methods and novel MRI contrast agent constructs are also
disclosed.
Inventors: |
Aryal; Santosh; (Manhattan,
KS) ; Pitchaimani; Arunkumar; (Via Morego, Genova,
IT) ; Nguyen; Tuyen DT; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Kansas State University Research Foundation |
Manhattan |
KS |
US |
|
|
Family ID: |
70853653 |
Appl. No.: |
17/296379 |
Filed: |
November 26, 2019 |
PCT Filed: |
November 26, 2019 |
PCT NO: |
PCT/US2019/063319 |
371 Date: |
May 24, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62772265 |
Nov 28, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/704 20130101;
A61P 35/00 20180101; A61K 47/6937 20170801; A61K 47/6901 20170801;
A61K 47/6923 20170801; A61K 49/1812 20130101; A61K 49/1878
20130101; A61K 49/1896 20130101; A61K 47/6929 20170801; A61K 9/1271
20130101; A61K 47/6911 20170801 |
International
Class: |
A61K 49/18 20060101
A61K049/18; A61K 9/127 20060101 A61K009/127; A61K 47/69 20060101
A61K047/69; A61P 35/00 20060101 A61P035/00; A61K 31/704 20060101
A61K031/704 |
Claims
1. A synthetic nanocarrier construct comprising a lipid-based
bilayer membrane infused with one or more NK-92 cell membrane
proteins.
2. The nanocarrier construct of claim 1, wherein said NK-92 cell
membrane proteins are surface protein receptors.
3. The nanocarrier construct of claim 2, wherein said surface
protein receptors are CD56, NKG2-D, NKp30, NKp44, CD16, or a
combination thereof.
4. The nanocarrier construct of claim 1, said lipid-based bilayer
membrane comprising a mixture of lipids and NK-92 cell membrane
phospholipids.
5. The nanocarrier construct of claim 4, wherein said lipids are
selected from the group consisting of phosphoethanolamines,
phosphatidylcholines, phosphoglycerols, phosphatidic acids,
Sphingolipids, Sphingomyelin, and combinations thereof.
6. The nanocarrier construct of claim 4, wherein said membrane
comprises at least two different types of lipids.
7. The nanocarrier construct of claim 4, further comprising
cholesterol and/or oleic acid.
8. The nanocarrier construct of claim 1, wherein said NK-92 cell
membrane proteins are located at the exterior surface of the lipid
bilayer, in the core of the lipid bilayer, and/or at the interior
surface the lipid bilayer.
9. The nanocarrier construct of claim 1, wherein said membrane
encapsulates a liquid-receiving interior space.
10. The nanocarrier construct of claim 9, further comprising one or
more active agents dispersed in said liquid-filled interior
space.
11.-12. (canceled)
13. The nanocarrier construct of claim 1, wherein said membrane
coats at least a portion of a solid core.
14. The nanocarrier construct of claim 13, wherein said solid core
is a polymeric or metal nanoparticle.
15.-16. (canceled)
17. The nanocarrier construct of claim 1, said membrane having an
exterior surface comprising one or more imaging agents or
detectable moieties.
18. (canceled)
19. The nanocarrier construct of claim 14, wherein said membrane
comprises an active agent incorporated therein.
20. (canceled)
21. A method of targeted delivery of an active/diagnostic/imaging
agent to a specific cell type or a region of a patient, said method
comprising administering a plurality of nanocarrier constructs of
claim 1 to said patient.
22. The method of claim 21, wherein said nanocarrier constructs
accumulate in and near an area of infection, inflammation, and/or
cancerous tissue in said patient.
23. (canceled)
24. The method of claim 21, wherein said nanocarrier constructs
fuse with said specific cell type and release an active agent
directly into the fused cell.
25. The method of claim 21, wherein said patient has is or
suspected of having cancerous or precancerous tissue or cancer
cells, wherein said nanocarrier constructs accumulate in said
cancerous or precancerous tissue or cancer cells.
26.-27. (canceled)
28. A diagnostic and/or therapeutic composition comprising a
plurality of nanocarrier constructs according to claim 1,
optionally dispersed in a pharmaceutically-acceptable carrier or
excipient.
29. An MRI imaging method for detecting cancerous or precancerous
tissue or cancer cells in a mammal comprising: (a) administering to
the mammal a plurality of nanocarrier constructs according to claim
1, said nanocarrier construct comprising at least one imaging
contrast agent, wherein said nanocarrier constructs accumulate in
cancerous or precancerous tissue or cancer cells in said mammal;
(b) locating said nanocarrier constructs in a region of interest in
the mammal suspected of having said cancerous or precancerous
tissue or cancer cells; (c) transmitting radio frequency pulses to
said region of interest; and (d) acquiring MR image data of the
region of interest, said MR image data comprising T.sub.1 data.
30.-35. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the priority benefit of U.S.
Provisional Patent Application Ser. No. 62/772,265, filed Nov. 28,
2018, entitled NANOCARRIER SYSTEMS FOR IMAGING AND DELIVERY OF
ACTIVE AGENTS, incorporated by reference in its entirety
herein.
BACKGROUND OF THE INVENTION
Field of the Invention
[0002] The present invention relates to nanocarriers comprising a
membrane or coating with natural killer (NK) cell membrane surface
proteins.
Description of Related Art
[0003] Biomimetic nanocarrier systems are a continuing area of
interest for targeted imaging and drug delivery. Inspired by
nature, these systems show promising biomedical applications, not
only as a biocompatible methodology in nanotechnology, but also it
mimics the function of natural biological materials. The biomimetic
approach has already proven the advantages of transforming natural
materials into functional materials ranging from drug delivery to
bio-sensors, for example, application of nanomaterials in cancer
theranostics. The advantages of incorporating biological materials
with synthetic materials include biocompatibility, resistivity,
cellular interaction, enhanced circulation half-life, and cellular
retention.
[0004] In the field of drug delivery, biomimetic nanoparticles
(NPs) provide an endogenous milieu for safer delivery of cargos
thereby reducing the toxicity of various organic and inorganic NPs.
Biomimetic nanoconstructs play a prominent role in reducing the
acceleration of immune response, which is the major clinical
pitfall during the administration of various organic and inorganic
NPs for biomedical ailments. In recent years, biomimetic and
synthetic nanostructures are combined to develop novel properties
to improve biomedical application.
[0005] For diagnostics and imaging, bioimaging modalities such as
MRI have advantages with excellent spatial resolution and
soft-tissue contrast for diagnosis and monitoring the therapeutic
response. The most common MRI contrast agents are gadolinium
(Gd)-based contrast agents (GBCA)(25-30% of MR scan includes GBCA).
Specific examples of GBCA include Gd-BOPTA (gadobenate dimeglumine,
Multihance.RTM.), GdDTPA (gadopentetate dimeglumine,
Magnevist.RTM.), Gd-EOB-DTPA (gadoxetic acid disodium,
Eovist.RTM.), MS325.RTM. (gadofosveset trisodium, Ablavar.RTM.),
etc. Despite advances in cancer bioimaging, early detection and
targeted bioimaging using MRI are highly challenging in most tumor
types. This is due to the lack of targeting moiety, short residence
time and free distribution to the extracellular spaces upon
injection.
[0006] Natural Killer (NK) cells are large granular lymphocytes
belongs to the innate immune system, whose major function is to
provide host defense against microbial infections and tumor
invasion by immunosurveillance of cell surfaces for the presence of
an abnormal expression of Major Histocompatibility Complex (MHC)
Class I molecules and cell stress markers. In peripheral blood
mononuclear cells, NK-cells contribute about 5-20%. Unlike T-cells
and B-cells, NK cells have the ability to target cancer cells
directly via inhibitory and activating receptors on its cell
surface and also can kill cancer cells without prior sensitization.
Its mechanism of cytotoxicity involves the release of membrane
disrupting protein (perforin) and a proteolytic enzyme (granzyme),
which cause lysis of target cells. Various mechanisms of NK cells
in targeting tumor include perforin/granzyme mediated cytotoxicity,
death receptor-mediated apoptosis, and interferon-.gamma. effector
function. Several studies have proved that NK cells are capable of
eliminating tumors in vitro and in vivo. Among various transformed
NK cell lines, NK-92 cells are an immortalized cell line derived
from a 50-year-old male patient with non-Hodgins lymphoma and
characterized by permanent IL-2 dependency. NK-92 is further
characterized by the presence of CD56 bright receptors and the
activated receptors like NKG2-D, NKp30, and NKp44 on its surface
for cytolytic functions. Unlike primary NK cells, NK-92 cells do
not have inhibitory receptors (KIR receptors), thus showing
superior cytotoxic activity against a broad range of tumors targets
compared to primary NK cells.
SUMMARY OF THE INVENTION
[0007] The present invention is broadly concerned with synthetic
nanocarrier constructs comprising a lipid-based bilayer membrane
infused with one or more NK-92 cell membrane proteins (e.g.,
surface receptor proteins derived from NK-92 membrane fragments).
The constructs are either hollow, liquid-filled constructs
(vesicles) or solid-core nanoparticles with a bilayer membrane
coating. Active agents, imaging agents, and/or detectable moieties
can be encapsulated within or conjugated to the membrane.
[0008] Also described herein are methods for targeted delivery of
an active/diagnostic/imaging agent to a specific cell type or a
region of interest of a patient. The methods generally comprise
administering a plurality of nanocarrier constructs according to
various embodiments to the patient.
[0009] Diagnostic and/or therapeutic compositions are also
disclosed, which comprise a plurality of nanocarrier constructs
according to various embodiments, optionally dispersed in a
pharmaceutically-acceptable carrier or excipient.
[0010] The present disclosure also concerns MRI imaging methods for
detecting cancerous or precancerous tissue or cancer cells in a
mammal. The methods generally comprise (a) administering to the
mammal a plurality of nanocarrier constructs according to various
embodiments which at least one MR imaging contrast agent, wherein
the nanocarrier constructs accumulate in cancerous or precancerous
tissue or cancer cells in the mammal; (b) locating the nanocarrier
constructs in a region of interest in the mammal suspected of
having said cancerous or precancerous tissue or cancer cells; (c)
transmitting radio frequency pulses to the region of interest; and
(d) acquiring MR image data of the region of interest which
comprises T.sub.1 data.
[0011] Also described herein are novel MRI contrast agent
nanocarrier constructs that comprise a synthetic nanocarrier
construct comprising a lipid-based bilayer membrane infused with
one or more NK-92 cell membrane proteins and at least one contrast
imaging agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0013] FIG. 1A. Schematic illustration of activated NK cells (NK-92
cells) and NK-92 cell membrane-derived fusogenic liposomes for
targeted tumor therapy.
[0014] FIG. 1B. NK cells recognize tumor cells by their
overexpressed surface stress markers and can induce its anti-tumor
potential by releasing cytotoxic granules. In NKsomes, doxorubicin
(DOX)-loaded NK cell membrane-camouflaged liposomes can recognize
tumor cells with the help of NK cell markers and fused with the
tumor cells more efficiently than normal cells and shows its
anti-tumor potential by the releasing the chemotherapeutic drug,
DOX.
[0015] FIG. 2A. Schematic illustration of the preparation of
NKsomes using sucrose density gradient centrifugation and an
extrusion method.
[0016] FIG. 2B. Transmission electron microscopic images of bare
liposomes and NKsomes.
[0017] FIG. 2C. Dynamic light scattering size distribution analysis
of NKsomes with different membrane:lipid ratio (1:100, 1:500 and
1:1000).
[0018] FIG. 2D. Zeta potential of isolated NK cell membrane, bare
liposomes, and NKsomes with different membrane content (1:100,
1:500 and 1:1000).
[0019] FIG. 2E. Estimation of lipid content from synthetic liposome
in NKsomes (1:500) analyzed using Thermogravimetric analysis.
[0020] FIG. 2F. FTIR analysis of NKsomes (1:500) showing functional
characterization of membrane proteins.
[0021] FIG. 2G. Dot-blot assay for the qualitative detection of
characteristic proteins in NK-92 cell extract and the isolated
NK-membranes using discontinuous sucrose gradient centrifugation.
Immunoblotting confirms the retention of cellular membrane protein
markers like CD-56, NKG-2D, and NKp30 and also confirms the absence
of nuclear material in the isolated NKM.
[0022] FIG. 3A. Fusogenic properties and protein characterization
of NKsomes. SDS-PAGE analysis of membrane proteins isolated from
NK-cell membrane and its presence in NKsomes.
[0023] FIG. 3B. Western blot analysis of isolated NKM and NKsomes
for its characteristics membrane proteins (CD 56, NKp30 &
NKG-2D).
[0024] FIG. 3C. Fluorescent resonance energy transfer (FRET) study
of NKM fused FRET NKsomes (NBD/RhB-labelled liposomes) with MCF-7
cells. Membrane fusion results in reduced FRET effect by increase
in fluorescent recovery of donor fluorophore (NBD .lamda.em=525
nm).
[0025] FIG. 3D. FACS analysis of immunostained MCF-7 cells for the
presence of NK cells surface protein markers (CD 56, NKp30 &
NKG-2D) before and after fusion with NKsomes.
[0026] FIG. 3E. In vitro cellular fusion of RhB labelled Nksomes in
MCF-7 cells after 3 h, immunostained for NK-92 cell marker (CD 56)
captured in CLSM.
[0027] FIG. 3F. Cellular uptake of NKsomes and NKM coated anionic
liposomes in MCF-7 cells after 3 h, immunostained with
FITC-anti-CD56.
[0028] FIG. 4A. Stability and drug loading/release characteristics
of NKsomes. Stability of NKsomes in PBS (pH=7) at 4.degree. C.
[0029] FIG. 4B. Serum stability of Bare liposomes, isolated NKM,
and NKsomes in 90% FBS analyzed spectrophotometrically at 560
nm.
[0030] FIG. 4C. Comparative DOX loading efficiency of NKsomes and
bare liposomes with various initial input concentrations of DOX
(25-200 .mu.g). Data represent mean f SD (n=3).
[0031] FIG. 4D. Comparative drug release characteristics of NKsomes
and Bare Liposomes in PBS (pH=7) and acetate buffer (pH 5.5) after
72 h incubation.
[0032] FIG. 5A. Targeting efficiency of NKsomes under flow
condition to MCF-7 cells and NHost cells. Flow passage analysis of
RhB-labeled NKsomes under flow with MCF-7 cells and its
time-dependent fluorescent accumulation.
[0033] FIG. 5B. Concentration-dependent fluorescence of
RhodamineB-labeled NKsomes in aqueous condition.
[0034] FIG. 5C. Differential cellular fusion of NKsomes and Bare
liposomes with MCF-7 and NHost cells under flow condition.
[0035] FIG. 5D. Confocal microscopic images of comparative cellular
fusion of NKsomes and Bare liposomes in MCF-7 and NHost, after 2 h
flow condition, immunostained for NK-cell membrane protein marker
(CD56).
[0036] FIG. 5E. Flow passage analysis of Rh-labelled bare liposomes
under flow condition with MCF-7 cells.
[0037] FIG. 5F. Flow passage analysis of Rh-labelled NKsomes and
bare liposomes under flow condition with NHost cells.
[0038] FIG. 6 In vitro immunogenicity assessment of NKsomes in
THP-1 cells after 24 h incubation. The immunostimulant effect of
engineered NKsomes was monitored by quantitative determination of
pro-inflammatory cytokines (IL-10, IL-6, and TNF-.alpha.) in THP-1
cells. Lipopolysaccharides (3 .mu.g/mL) were used as a positive
control. Data represent Mean f Standard Deviation (n=3).
***P<0.001, *P<0.5 by compared to control cells.
[0039] FIG. 7A. In vitro therapeutic efficacy/cytotoxicity of
different concentration of DOX@NKsomes (0.5-15 .mu.M) in MCF-7
after 24 hr incubation.
[0040] FIG. 7B. In vitro therapeutic efficacy/cytotoxicity of
different concentration of DOX@NKsomes (0.5-15 .mu.M) in NHost
after 24 h incubation.
[0041] FIG. 7C. Biocompatibility of NKsomes in MCF-7 cell after 24
h incubation.
[0042] FIG. 7D. Cell cycle histogram of Free DOX and DOX@NKsomes
(DOX concentration=5 .mu.M) treated cells showing different cell
cycle phase distribution.
[0043] FIG. 7E. Percentage of G1 cell cycle phase distribution of
MCF-7 and NHost cells treated with Free DOX and DOX@NKsomes after
12 h incubation. ***P<0.001, **P<0.01 compared to control
cells.
[0044] FIG. 7F. Percentage of G2 cell cycle phase distribution of
MCF-7 and NHost cells treated with Free DOX and DOX@NKsomes after
12 h incubation. ***P<0.001, **P<0.01 compared to control
cells.
[0045] FIG. 8A. Blood retention of DiR-loaded NKsomes in MCF-7
bearing tumor mice after single intravenous injection to evaluate
the circulation half-life of NKsomes through the two-compartmental
model. Insert shows the non-linear elimination model of
NKsomes.
[0046] FIG. 8B. Bio-distribution of NKsomes in MCF-7 bearing tumor
mice after 24 h. DiR-labelled NKsomes were injected intravenously
through tail vein and after 24 h major organs and tumors were
collected, homogenized, and quantified by measuring the DiR signals
using NIR spectrophotometer. Data represent mean f SD, n=3.
[0047] FIG. 8C. Representative immunofluorescence image of tumor
tissues showing the bioaccumulation of NKsomes, as illustrated by
the FITC fluorescence signals of NK-cell marker, CD-56. Nuclei were
further stained with DAPI. Scale bar=50 .mu.m.
[0048] FIG. 9A. In vivo anti-tumor effect of DOX-loaded NKsomes
(DOX@NKsomes) against MCF-7 derived solid tumor model in NU/NU
mice. Change in tumor volume of mice treated with free DOX (5
mg/kg), DOX-loaded NKsomes (equivalent DOX concentration, 5 mg/kg)
along with bare NKsomes (10 mg/kg) and untreated controls.
DOX@NKsomes inhibits tumor growth in MCF-7 tumor-bearing
immunodeficient NU/NU nude mice.
[0049] FIG. 9B. Body weight changes of tumor bearing mice treated
with free DOX (5 mg/kg), DOX-loaded NKsomes (equivalent DOX
concentration, 5 mg/kg) along with bare NKsomes (10 mg/kg) and
untreated controls.
[0050] FIG. 9C. Quantified tumor weight of different treatment
groups (Untreated control, Bare NKsomes, Free DOX and DOX@NKsomes)
at the end of the therapeutic study. Data represent mean f SD
(*P<0.05, **P<0.01, ***P<0.001 compared to untreated
control, n=3).
[0051] FIG. 9D. Tumor image of control, NKsomes, Free DOX and
DOX@NKsomes treated animals at the end of the therapeutic
study.
[0052] FIG. 9E. Representative tumor tissue sections of untreated,
NKsomes, Free DOX, and DOX@NKsomes treated mice subjected to
confocal analysis for the distribution of DOX and NKsomes. Tissues
were immunostained with FITC-anti-CD-56 for CD-56, and the nucleus
was stained using 4', 6-Diamidino-2-phenylindole, dihydrochloride
(DAPI). Scale bar=50 .mu.m.
[0053] FIG. 9F. Representative dot-blot analysis of tumor tissue of
DOX@NKsome treated animals for the qualitative determination of its
characteristics proteins (CD 56, NKG2D, and MIC-A/B).
[0054] FIG. 10A. Schematic illustration of the preparation of
biomimetic nanoconstructs (BNc) made up of natural killer cell
membrane isolated using sucrose gradient differential
centrifugation. BNc were prepared using simple extrusion technique,
where PLGA nanoparticles and Gd-lipids were extruded with isolated
NKM.
[0055] FIG. 10B. DLS size of the BNc before and after NKM
coating.
[0056] FIG. 10C. Transmission electron microscopic image of the
prepared BNc, insert shows the magnified image of the BNc with
distinct biomolecular corona (NKM and Gd-lipid).
[0057] FIG. 11. Protein characterization of BNc. (A) SDS-PAGE
analysis of protein bands in NK-92 cell extract, isolated NKM, and
the BNc. (B) Western blot analysis of BNc for its characteristic
protein, CD 56, NKG-2D, and NKp30.
[0058] FIG. 12A. Stability of BNc in PBS (pH=7.4) at 4.degree.
C.
[0059] FIG. 12B. Serum stability of BNc and bare PLGA-NP in 90% FBS
at 37.degree. C.
[0060] FIG. 12C. Gd.sup.3+ loading efficiency of BNc with different
initial input concentrations of Gd-lipid (50-400 .mu.g).
[0061] FIG. 12D. Gd.sup.3+ release characteristics of BNc in PBS
(pH=7.4) at 37.degree. C.
[0062] FIG. 13A. T.sub.1 recovery curve of BNc in the presence and
the absence of NKM, showing r1=5.0 mM-1s-1.
[0063] FIG. 13B. T.sub.1 contrast phantoms of different
concentration of BNc (0.05 mM-0.4 mM).
[0064] FIG. 13C. T.sub.1 recovery curve of different concentrations
of BNc (0.05 mM-0.4 mM) acquired at 14.1 T.
[0065] FIG. 14A. Confocal laser scanning z-stack micrograph of
cellular uptake of BNc in MCF-7 cells after 3 h of incubation,
immunostained with FITC-anti-CD 56.
[0066] FIG. 14B. FACS analysis of quantitative cellular uptake of
BNc in comparison with bare PLGA-NP after 6 h incubation.
[0067] FIG. 14C. Biocompatibility of different concentration of BNc
(10-150 .mu.g) in MCF-7 cells after 24 h incubation
[0068] FIG. 15. Confocal laser scanning micrograph of cellular
uptake of BNc in MCF-7 cells after 3 h of incubation, immunostained
with FITC-anti-CD 56.
[0069] FIG. 16. In vitro immunogenicity of BNc in THP-1 cells
assessed by evaluating the pro-inflammatory cytokines (IL-1.beta.,
IL-6, and TNF-.alpha.) release after 24 h incubation.
[0070] FIG. 17A. Time dependent live animal imaging of MCF-7 tumor
bearing NU/NU mice after intravenous injection of DiR-labelled BNc
(10 mg/kg). Images were recorded prior to injection and after 3 h,
6 h, 12 h and 24 h, respectively.
[0071] FIG. 17B. Bio-accumulation of BNc and control PLGA
nanoparticles in tumor bearing mice after 24 h post-injection.
[0072] FIG. 17C. Comparative tumor accumulation of BNc and Bare
PLGA nanoparticles in MCF-7 tumor bearing NU/NU mice after 24 h
post-injection.
[0073] FIG. 18A. Circulation half-life of BNc in MCF-7 tumor
bearing NU/NU mice over 72 h time.
[0074] FIG. 18B. Bio-distribution profile of BNc after single
intravenous tail-vein injection in MCF-7 tumor-bearing NU/NU
mice.
[0075] FIG. 18C. Ex vivo T.sub.1 weighted magnetic resonance image
slices of NU/NU mice bearing MCF-7 tumor after 2 h post-injection.
Slice thickness: 0.5 mm. Color circles indicate the standard
controls; red: water, yellow: 18 .mu.M Magnevist.RTM., blue: 37
.mu.M Magnevist.RTM..
[0076] FIG. 19. Ex vivo MRI images of MCF-7 tumor bearing
mouse.
[0077] FIG. 20. Transmission electron microscopic images of: (A)
citrate gold Nanoparticles (AuNPs) and (B) Natural Killer cell
membrane (NKM) coated AuNPs.
[0078] FIG. 21. Spectrophotometric characterization of AuNPs before
and after NKM coating showing surface plasmon resonance (SPR) band
at 525 nm. Stable SPR band at 525 nm further suggest stabile and
intact size properties after coating.
[0079] FIG. 22. Zeta potential measurement. Citrate stabilized
AuNPs are negatively charged (-40 mV) where are NKM coated NPs are
-18 mV. This change is surface charge reveled the formation of NKM
coating over AuNPs, which is similar to that of cell surface.
[0080] FIG. 23. Hydrodynamic size of AuNPs before and after coating
with NKM determined by dynamic light scattering. After coating
hydrodynamic size increased from 30 nm to 70 nm, which is due to
hydration layer and larger cellular protein molecules on the
surface.
[0081] FIG. 24A. NKM coated AuNPs internalization in MCF-7 cells.
Black arrow in inset 1, 2, and 3 (in red) shows endosomal membrane
rupture.
[0082] FIG. 24B. Citrated stabilized AuNPs internalization. Inset 1
and 2 (in black) shows intact endosomal membrane and NP cell
membrane interaction, respectively.
DETAILED DESCRIPTION
[0083] In more detail, described herein are nanocarrier constructs
comprising a lipid-based bilayer membrane infused with one or more
NK-92 cell membrane proteins. The lipid-based bilayer membrane
comprises a plurality of lipids having respective hydrophilic heads
and two hydrophobic tails. Such lipids will spontaneously (or
automatically) self-assemble into a bilayer morphology. The
membrane is preferably heterogenous comprising at least two
different lipids.
[0084] In some embodiments, the membrane coats a solid core. In
other embodiments, the membrane encloses a hollow liquid-receiving
space.
[0085] NK-92 cell membrane proteins that can be incorporated into
the lipid-based bilayer membrane include surface protein receptors,
such as CD56, NKG2-D, NKp30, NKp44, CD16, and the like. The
infusion of NK-92 cell membrane can be driven by electrostatic
and/or hydrophobic interaction of the NK-92 cell membrane
components (protein and NK-92 membrane phospholipid fragments) with
the lipid bilayer components. The infused NK-92 membrane
phospholipid becomes a part of the lipid bilayer, meanwhile the
NK-92 cell membrane proteins can locate at the exterior surface of
the lipid bilayer (facing the external environment), in the middle
of the lipid bilayer, or at the interior surface the lipid bilayer
(facing the core). The membrane can be permeable, non-permeable, or
semi-permeable, and is preferably semi-permeable. Exemplary lipids
for use in forming the membrane include natural phospholipids and
modified phospholipids, such as phosphoethanolamines,
phosphatidylcholines, phosphoglycerols, phosphatidic acids,
Sphingolipids, Sphingomyelin, and the like, such as
1,2-dioleoyl-3-trimethylammonium-propane (DOTAP),
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),
L-.alpha.-phosphatidylcholine (Egg-PC),
1,2-Distearoyl-sn-glycero-3-phosphoglycerol (DSPG),
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), DSPE
conjugated 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic
acid-Gd (DSPE-DOTA-Gd), and combinations thereof. Preferably, the
membrane is heterogeneous, comprising at least two different
lipids. Other membrane constituents can include cholesterol, oleic
acid, hydrophobic drug molecules, imaging agents, detectable
moieties, and the like. The nanocarriers have an average particle
size of at least 100 nm, but less than 1,000 nm, preferably less
than 500 nm, and more preferably less than about 150 nm. As used
herein, the "particle size" refers to the maximum
surface-to-surface dimension of the body, such as the diameter in
the case of substantially spherical bodies.
[0086] In one or more embodiments, the lipid bilayer membrane
encapsulates a liquid-receiving interior space (i.e., hollow core),
similar to a liposome. Thus, nanocarriers in such embodiments are
substantially spherical vesicles having at least one lipid bilayer,
and a hollow core capable of trapping active agents and other
molecules in their liquid-filled interior space. In such
embodiments, hydrophobic residues of the lipids face inward
defining the inner "core" of the vesicle membrane bilayer, while
the hydrophilic heads face outward from the core of the membrane
bilayer towards the internal and external vesicle environments and
thereby define the interior and exterior surfaces of the membrane.
In other words, the hollow core of the vesicle is filled with
liquid (e.g., aqueous solutions such as normal (n.) saline (-0.9%
NaCl), phosphate buffered saline (PBS), and/or sterile water (DAW),
oil-in-water or water-in-oil emulsions) in which the active
agents/molecules are dispersed, such that the liquid and active
agents/molecule are entrapped by the lipid-based bilayer membrane
infused with one or more NK-92 cell membrane proteins. Such
nanocarrier vesicles are also referred to herein as "NKsomes" in
some embodiments of the invention. Advantageously, NKsomes are
designed to provide biomimetic cloaking of the synthetic
nanocarrier and encapsulated active agent using a membrane
camouflage. The NKsomes are characterized by NK cell
membrane-associated targeting proteins on the membrane surface,
which have been derived from NK cell membrane fragments.
[0087] With its excellent biocompatibility, NKsomes show a higher
affinity towards cancer/tumor cells than normal cells, and exhibit
enhanced tumor homing efficiency in vivo with an extended plasma
residence time of 18 h. The NKsomes are also fusogenic, meaning
they will fuse with the target (tumor) cells and release their
payload (i.e., the contents of their liquid core) directly into the
fused cells. Thus, NKsomes are particularly useful for targeted
tumor therapy and delivery of chemotherapeutic agents in a targeted
manner, with minimal off-target effects.
[0088] In one or more alternative embodiments, the lipid bilayer
membrane encapsulates or coats at least a portion of a solid
nanoparticle core. That is, each nanoparticle is individually
encapsulated or coated by a corresponding membrane. The term
"nanoparticle" as used herein refers to submicron-sized colloidal
particles. In general, such nanoparticles will have a particle size
of at least 100 nm, but less than 1,000 nm, preferably less than
500 nm, and more preferably less than about 150 nm. In one or more
embodiments, the nanoparticles are polymeric nanoparticles, which
may be formed from a single polymer, copolymers, or mixtures of two
or more polymers. Non-limiting examples of polymers that can be
used for the nanoparticles include poly(glycolic acid), poly(lactic
acid), poly(lactic-co-glycolic acid), poly(caprolactone),
poly(ortho esters), poly(alkyl cyanoacrylates), poly(sebacic acid),
poly(adipic acid), poly(terphthalic acid), poly(.gamma.glutamic
acid), poly(L-lysine), poly(.beta.-amino esters),
poly(phosphoesters), polycarbonates, polyvinylpyrrolidone
ethylcellulose, poloxamer, polyamidoamine, polyglycerol, sodium
pyrrolidone carboxylate, chitosan, and the like.
[0089] In one or more embodiments, metal nanoparticles can be used
for the nanoparticle core. Exemplary metal nanoparticles comprise a
metal or metal alloy of one or more metals selected from the group
consisting of iron, gold, manganese, oxides thereof, and
combinations thereof.
[0090] Thus, nanocarriers in such embodiments are substantially
spherical particles having at least one lipid bilayer membrane as a
coating, and a solid nanoparticle core, wherein active agents can
be encapsulated/conjugated with the nanoparticle and/or the
NK-infused lipid-based membrane. As with NKsomes, these
nanoparticle carriers are characterized by NK cell
membrane-associated targeting proteins on the bilayer membrane
surface, such that these coated nanoparticle carriers have a high
affinity towards cancer/tumor cells and enhanced tumor homing
efficiency (i.e., are tumor trophic).
[0091] Further, in one or more embodiments, the membrane can
further include one or more contrast media, imaging agents, or
detectable moieties (e.g., dyes) for imaging and/or diagnostics,
such as gadolinium, fluorescent dyes (e.g., Near infrared dye
(NIR-dye) of different colors, such as Alexa Fluor.RTM., Cy.RTM.,
and IR.RTM. Dyes), radioactive isotopes (e.g., Copper-64,
fluorine-18 (FDG-18), Technetium-99, zirconium (Zr-95, Zr-88,
Zr-89), iodinated contrast agents), and the like. The contrast
agents can be pre-conjugated to the lipids used to form the bilayer
membrane, such that they will be integrally formed with the bilayer
membrane, and be present on the interior and/or exterior surfaces
of the membrane (i.e., at the hydrophilic heads). These moieties
can be attached to the hydrophilic component of a membrane lipid,
which will preferably predominately occupy the outer layer of the
bilayer membrane, thus presenting the moiety on the exterior
surface of the membrane after formation. Imaging agents can also be
conjugated to the membrane surface after formation around the
nanoparticle core. When the appropriate contrast agent is used, the
magnetic properties of the coated nanoparticles can be tunable from
2.1.+-.0.17 to 5.3.+-.0.5 mM.sup.-1s.sup.-1 (e.g., under 14.1 T) by
adjusting the concentration of imaging agent on the surface of the
nanocarriers. The current data was obtained using 14.1 T, but it
will be appreciated that various magnetic strengths may be used,
depending upon the particular MRI machine. The coated nanoparticle
nanocarriers have a circulation half-life of about 9.5 h, and a
high biodistribution in tumor tissues (10% of injected dose).
[0092] Active agents that can be encapsulated and/or conjugated to
the nanocarriers in various embodiments include both hydrophobic
and hydrophilic agents. Examples include among other things, drugs
(small molecule compounds, macromolecules) and other therapeutic
molecules such as antibiotics, bioactive compounds, nutraceuticals,
enzymes and other proteins and peptides, DNA and RNA (e.g.,
recombinant nucleic acids, RNA oligomers, DNA plasmids), prodrugs,
and the like. Chemotherapeutic agents and other anticancer agents
are particularly suited for use in the invention.
[0093] Non-limiting examples of active agents include:
TABLE-US-00001 Hydrophobic compounds Hydrophilic compounds (water
insoluble) (water soluble) Anticancer drugs Paclitaxel Doxorubicin
Retinoic acid Daunorubicin Docetaxel Edelfosine Cisplatin
Gemcitabine Etoposide Vincristine Curcumin Oxiplatin Etoposide
Irinotecan Methotrexate Temsirolimus 5-Fluorouracil carmustine
Carboplatin Antibiotics Cephalosporins Penicillins Quinolones
Tetracyclines Macrolides Lincomycins Sulfonamides Glycopeptides
Carbapenems Aminoglycosides Biologics Proteins Nucleic acids
Various classes of peptides Aptamers Cells and cellular components
such as: extracellular vesicles/exosomes from immune cells,
diseased cells, or healthy cells of various types, such as stromal
cells.
Excipients and other carriers or liquid solutions may also be
included inside the vesicle nanocarriers, along with adjuvants
(e.g., alum, aluminum hydroxide, aluminum phosphate, calcium
phosphate hydroxide, detergents, such as Quil A, and other
saponins, mineral oils, squalene, Freund's complete or incomplete
adjuvants), and the like. Other than therapeutics, various
bioimaging contrast agents (MRI contrast agents, such as Gd-based
agents) can also encapsulated/conjugated within the synthetic
nanoparticles aiming to prolong circulation half-life and enhance
tumor accumulation.
[0094] These nanocarriers can be used in pharmaceutically
acceptable compositions for delivering the
active/diagnostic/imaging agents and can be administered
intravenously, subcutaneously, intramuscularly, orally,
intraperitoneally, or via inhalation to a subject. Methods of
targeting delivery of an active/diagnostic/imaging agents to a
specific cell type or a region of a patient are also contemplated
herein. In one or more embodiments, the composition comprises an
effective amount of nanocarrier dispersed in a
pharmaceutically-acceptable carrier or excipient. A
pharmaceutically-acceptable carrier or excipient would naturally be
selected to minimize any degradation of the nanocarrier and to
minimize any adverse side effects in the subject, cells, or tissue,
as would be well known to one of skill in the art.
Pharmaceutically-acceptable ingredients include those acceptable
for veterinary use as well as human pharmaceutical use. Exemplary
carriers and excipients include aqueous solutions such as n.
saline, PBS, and/or DAW, oil-in-water or water-in-oil emulsions,
and the like. As used herein, an "effective" amount refers to the
amount of the nanocarrier that will elicit the biological or
medical response of a tissue, system, animal, or human that is
being sought by a researcher or clinician, and in particular elicit
some desired therapeutic/diagnostic/imaging effect. One of skill in
the art recognizes that an amount may be considered effective even
if the condition is not totally eradicated but improved
partially.
[0095] In certain embodiments, the nanocarrier may further be
administered in combination with additional secondary agents,
including anti-inflammatory agents, immunomodulators, and
antimicrobial agents, such as antivirals, antibiotics,
anti-fungals, anti-parasitics, and the like.
[0096] In one or more embodiments, nanocarrier vesicles can be
prepared by hydrating NK-92 membrane fragments with selected lipids
in a suitable buffer or solvent system, along with any imaging
agents or detectable moieties, and/or active agents followed by
extrusion through a membrane filter. The protocol can be optimized
by first forming a lipid thin film, followed by hydration. Further,
the solution can be sonicated to homogeneously intermix the
components before extrusion. It will be appreciated that the
imaging agents and/or detectable moieties could also be conjugated
to the vesicle surface after extrusion if desired. Likewise,
certain active agents could also be conjugated to the vesicle
surface after extrusion if desired. In one or more embodiments,
NK-92 cell membrane fragments are obtained from activated NK-92
cells (i.e., NK-92 cells that have been stimulated, such as with
cytokines, IFN-.gamma., IL-2, IL-4, IL5, IL-6, IL8, IL-10, IL-17,
TGF-.beta., and TNF-.alpha., and the like). In one or more
embodiments, methods of the invention involve ex vivo expansion and
activation of the NK-92 cells. Methods for expansion and activation
of NK cells are described in the literature. The cells are first
lysed and the membrane fragments isolated, such as by gradient
centrifugation. Isolated membrane fragments can be lyophilized for
storage until use and/or suspended in buffer. The NK-92 cell
membrane fragments are then mixed with the other components (e.g.,
lipids, active agents, etc.) for extrusion. Alternatively, the
NK-92 cell membrane fragments can be co-extruded with pre-formed
liposomes (which have already been loaded with active agents and/or
imaging agents) to integrate the NK-92 cell membrane fragments (and
associated NK-92 membrane proteins) in the lipid bilayer.
[0097] In one or more embodiments, solid-core nanocarriers can be
prepared by hydrating NK-92 membrane fragments with selected lipids
and nanoparticles in a suitable buffer or solvent system, along
with any imaging agents or detectable moieties, and/or active
agents followed by extrusion through a membrane filter. As noted,
polymeric and/or metal nanoparticles can be used. The NK-92
membrane fragments can be isolated as described above. The lipids
can be conjugated with active agents and/or imaging agents before
extrusion. Alternatively, these agents can be conjugated onto the
solid-core nanocarrier surface after extrusion. Alternative methods
for forming lipid bilayers for nanovesicles and/or solid core
nanocarriers include sonication, thin film evaporation, hydration,
nanoemulsion, extrusion, and the like.
[0098] Additional advantages of the various embodiments of the
invention will be apparent to those skilled in the art upon review
of the disclosure herein and the working examples below. It will be
appreciated that the various embodiments described herein are not
necessarily mutually exclusive unless otherwise indicated herein.
For example, a feature described or depicted in one embodiment may
also be included in other embodiments, but is not necessarily
included. Thus, the present invention encompasses a variety of
combinations and/or integrations of the specific embodiments
described herein.
[0099] As used herein, the phrase "and/or," when used in a list of
two or more items, means that any one of the listed items can be
employed by itself or any combination of two or more of the listed
items can be employed. For example, if a composition is described
as containing or excluding components A, B, and/or C, the
composition can contain or exclude A alone; B alone; C alone; A and
B in combination; A and C in combination; B and C in combination;
or A, B, and C in combination. The present description also uses
numerical ranges to quantify certain parameters relating to various
embodiments of the invention. It should be understood that when
numerical ranges are provided, such ranges are to be construed as
providing literal support for claim limitations that only recite
the lower value of the range as well as claim limitations that only
recite the upper value of the range. For example, a disclosed
numerical range of about 10 to about 100 provides literal support
for a claim reciting "greater than about 10" (with no upper bounds)
and a claim reciting "less than about 100" (with no lower
bounds).
EXAMPLES
[0100] The following examples set forth methods in accordance with
the invention. It is to be understood, however, that these examples
are provided by way of illustration and nothing therein should be
taken as a limitation upon the overall scope of the invention.
Example 1
Natural Killer Cell Membrane Infused Biomimetic Liposomes for
Targeted Tumor Therapy
[0101] In the present study, we designed the biomimetic
nanoconstructs made up of NK cell membrane infused fusogenic
liposomes (NKsome) for targeted drug delivery to the tumor as
demonstrated in the proposed mechanism (FIG. 1). An activated NK
cell membrane with receptor proteins was isolated from the NK-92
cells (ATCC CRL-2407) and membrane extruded with the fusogenic
liposome to form "NKsomes." In vitro tumor targeting ability of the
fusogenic NKsome was investigated against normal human osteoblast
(NHost) and Human breast cancer cells (MCF-7). Further, the
chemotherapeutic drug, doxorubicin (DOX) was encapsulated into the
aqueous core of NKsome and investigated its tumor homing ability
and anti-tumor efficacy against MCF-7 induced solid tumor model in
NU/NU mice.
[0102] 1. Materials and Methods
1.1. Chemicals and Antibodies
[0103] Lipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)
and 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt)
(DOTAP), were purchased from Avanti Polar Lipid Inc. (Alabaster,
Ala., USA). Cholesterol was purchased from Fisher. Chemotherapeutic
Drug, DOX was purchased from LC Laboratories (Woburn, Mass., USA).
Primary antibodies like NKG2-D, NKp30 and Pan-cadherin were
purchased from Santa Cruz Biotechnology. NCAM (CD56) and Secondary
HRP-linked anti-mouse IgG antibody were procured from Cell
Signaling. Fluorophore tagged antibodies like FITC Mouse anti-Human
CD56, PE-Cy.TM.7 Mouse Anti-Human CD314/NKG2D and Alexa Fluor.RTM.
647 Mouse Anti-Human CD337 (NKp30) and APC Mouse IgG1, .kappa.
Isotype Control were purchased from BD Bioscience. All other
reagents and chemicals were of analytical grade.
1.2. Cell Lines and Tumor Models
[0104] Human Natural Killer cells, NK-92 (ATCC.RTM. CRL-2407.TM.)
were procured from ATCC, Manassas, USA, and the cells were
maintained in Alpha Minimum Essential medium without
ribonucleosides or deoxyribonucleosides supplemented with 2 mM
L-glutamine, 1.5 g/l sodium bicarbonate, 0.02 mM folic acid, 0.1 mM
2-mercaptoethanol, 0.2 mM inositol, 200 U/ml recombinant IL-2 and
12.5% horse serum and 12.5% fetal bovine serum. Normal Human
Osteoblast cells, NHost was procured from Lonza, Inc and maintained
in OGM.RTM. Bullet Kit supplemented with 10% (v/v) fetal bovine
serum (FBS) and penicillin/streptomycin (100 ug/ml) and maintained
at 37.degree. C. in 5% CO.sub.2 environment. The human breast
cancer cell line MCF-7 (ATCC.RTM. HTB-22.TM.) was procured from
ATCC, Manassas, USA, and the cells were maintained in Dulbecco's
Modified Eagle's Medium (DMEM) supplemented with 10% (v/v) fetal
bovine serum (FBS) and penicillin/streptomycin (100 ug/ml) and
maintained at 37.degree. C. in 5% CO.sub.2 environment. Human
peripheral blood monocyte THP-1 cells were gifted by Dr. Massaki
Tamura, Kansas State University, USA and maintained in RPMI medium
with 10% (v/v) fetal bovine serum (FBS) and penicillin/streptomycin
(100 ug/ml) and maintained at 37.degree. C. in 5% CO.sub.2
environment.
[0105] Six-week-old female NU/NU nude mice were procured from
Charles River Laboratories International, Inc and used for the
study after 10 days acclimatization. All animal experiments and
protocols were approved by Institutional Animal Care and Use
Committee (IACUC) and Institutional Biosafety Committee (IBC),
Kansas State University, Manhattan. For tumor models,
1.times.10.sup.6 MCF-7 cells in saline were injected subcutaneously
into the hind rear flank region of the mice and the tumor growth
were monitored periodically.
1.3. Extraction of NK Membranes (NKM)
[0106] Natural killer cell, NK-92 cells were grown to 80%
confluence in multiple T-75 culture flask (.about.3.times.10.sup.8
cells) and harvested, washed in 1.times.PBS thrice by centrifuging
at 500 g for 5 min. The purified cell pellet was suspended in
Homogenization buffer (10 mM Tris-HCl, 1 mM KCl, 25 mM Sucrose, 1
mM MgCl.sub.2, 2 mM PMSF, 200 .mu.g/mL Trypsin-chymotrypsin
Inhibitor, 10 .mu.g/mL DNase and 10 .mu.g/mL RNase) and homogenized
in ice for 5 min (20 s pulse and 30 s in between pulses). The
homogenized mixture was collected under the ice-cold condition and
pooled over the discontinuous sucrose gradient (55%, 40% and 30%
(w/v) sucrose in 0.85% saline) in polycarbonate tubes. The sampled
gradients were ultra-centrifuged in a Beckman SW 27 rotor at 28,000
g for 30 min at 4.degree. C. The membrane fraction at 30% to 40%
interface was collected in a clean tube. For purification,
collected membrane fractions were diluted with an excess of normal
saline and ultracentrifuged in a Beckman SW 27 rotor at 28,000 g
for 1 h at 4.degree. C., and analyzed for protein characterization
using the dot-blot technique. The isolated membranes were
lyophilized, weighed, quantified for the protein concentration by
rehydrated in PBS (pH 7.4) and stored at 4.degree. C.
1.4. Preparation of Fusogenic NKsomes
[0107] Fusogenic NKsomes were prepared using a standard liposome
fabrication membrane extrusion technique. In brief, cationic
fusogenic liposome was prepared using DOTAP:DOPE:cholesterol
dispersed in chloroform (molar ratio of 47:44:9) using thin-film
hydration technique. The evaporated dried film was hydrated with
1.times.PBS (pH 7.4) and incubated at 40.degree. C. for 30 min,
mixed vigorously and further sonicated to obtain a clear suspension
of lipids. The liposomal suspensions were further extruded with
polycarbonate membrane filter with different pore sizes (1 .mu.m
and 200 nm), and the fusogenic liposome (bare liposome) was stored
at 4.degree. C. For NKsomes, bare liposome was further extruded
with 200 nm, in the presence of varying concentration of isolated
NK membrane (NKM) to make the overall protein:lipid ratio of
1:1000, 1:500 and 1:100 (by weight) namely NKsomes-A, NKsomes-B,
and NKsomes-C, respectively, and purified using Sephadex G-50
column (GE healthcare).
TABLE-US-00002 TABLE 1 Weight ratios of NKsome formulations.
Protein:Lipid Mohr ratio (%) ratio (by Formulation DOPE DOTAP
Cholesterol weight) Bare LIPO 47 44 9 0:1 NKsomes A 47 44 9 1:100
NKsomes B 47 44 9 1:500 NKsomes C 47 44 9 1:1000
The purified NKsomes are stored at 4.degree. C. until use. The
conventional anionic liposome was prepared using
DSPG:DSPG-PEG-succinyl:cholesterol in the molar ratio of 55:40:15
for control experiments. For Rhodamine labelled-experiments, 20
.mu.g of L-.alpha.-Phosphatidylethanolamine-N-(lissamine rhodamine
B sulfonyl) (Ammonium Salt) (Egg Liss Rhod PE) was included in the
NKsome formulation.
1.5. Characterization of Fusogenic NKsomes
[0108] The hydrodynamic size and zeta potential of the bare and DOX
encapsulated NKsomes were characterized by Dynamic light scattering
analysis (Malvern ZSP). The surface morphology of the liposome was
studied using Transmission electron microscope (FEI Technai G2
Spirit BioTWIN). Fourier transform infrared spectroscopic analysis
of the lyophilized NKsomes samples was analyzed using Nicolet.TM.
iS.TM. 50 FT-IR Spectrometer (Thermo Fisher). Protein
quantification in all samples was done using Bradford Assay. For
SDS-PAGE analysis, whole cell lysate, NKM, NKsomes samples were
prepared at a protein concentration of 300 .mu.g/mL. NKsomes were
collected by centrifugation at 12,000 rpm for 15 min and
redispersed in gel loading dye. All samples were heated at
90.degree. C. for 5 min, and 20 .mu.L of samples were loaded into
wells of 4-20% Mini-PROTEAN.RTM. TGX Protein Gels and stained using
Coomassie Brilliant Blue. For Western blot analysis, proteins were
transferred to PVDF membrane by the wet-blot method, and the
membranes were treated with primary antibodies for CD-56 (Cell
Signaling), NKG2-D (Santa Cruz), NKp30 (Santa Cruz) and
Pan-cadherin (Santa Cruz) along with HRP-conjugated anti-mouse IgG
secondary antibody (Cell Signaling). The blotted films were further
developed using SignalFire.TM. ECL Reagent (Cell signaling) and
imaged for chemiluminescence signal under Bio-imager (Kodak). For
the confocal study, 50,000 cells/well in 8-chamber slides were
treated with NKsomes and conventional anionic liposomes with NKM
coating (50 .mu.g/mL) for 3 h at 37.degree. C. After incubation,
excess media was removed, washed twice and samples were further
immunostained as described above and observed under Confocal Laser
Scanning Microscope (Carl Zeiss, LSM-700).
1.6. Fusogenic Property of NKsomes
[0109] The fusogenic property of the NKsomes was investigated using
fluorescent resonance energy transfer (FRET) study. In brief,
fusogenic FRET liposome was prepared by incorporating FRET
fluorophore lipids, an electron donor
(l-.alpha.-Phosphatidylethanolamine-N-(4-nitrobenzo-2-oxa-1,3-diazole)
(Ammonium Salt), 0.3 mole % and an electron acceptor
(1-.alpha.-Phosphatidylethanolamine-N-(lissamine rhodamine B
sulfonyl) (Ammonium Salt), 0.7 mole % into the NKsome formulation.
For fusion study, 50 .mu.L FRET liposome (1 mg/mL) were allowed to
incubate with 5.times.10.sup.3 MCF-7 cells in a 96-well plate at
room temperature for 5 min, and the cell samples were analyzed
spectrofluorimetrically by exciting sample at 470 nm and measuring
the emission spectrum between 500 nm and 700 nm. For cellular
fusion of NKsomes using fluorescent activated cell sorting (FACS),
1.times.10.sup.6 MCF-7 cells grown in the T25 flask were treated
with 500 .mu.g/mL of NKsomes and incubated for 3 h. After
incubation, excess media was removed, washed twice and trypsinized
and samples were immunostained with FITC Mouse anti-Human CD56,
PE-Cy.TM.7 Mouse Anti-Human CD314/NKG2D and Alexa Fluor.RTM. 647
Mouse Anti-Human CD337 (NKp30) and APC Mouse IgG1, .kappa. Isotype
Control and analyzed in BD FACS Calibur. For the confocal study,
NKsomes and conventionally treated cell samples were immunostained
and observed under Confocal Laser Scanning Microscope (CLSM) (Carl
Zeiss, LSM-700).
1.7. Drug Loading and Release Assay
[0110] For DOX encapsulation, NKsomes were prepared as described
earlier, in which the dried lipid cakes were directly hydrated with
calculated DOX (2.5%, 5%, 10%, 15% and 20% by total lipid weight)
in PBS, incubated at 50.degree. C. for 5 min and probe sonicated
for 1 min (20 s pulse). The liposomal dispersions were further
extruded in the presence of NKM (1:500) by using a Millipore
membrane filters with different pore sizes (1 .mu.m and 200 nm) and
the excess unbound DOX were column purified in Sephadex G-50
column. The drug loading and encapsulation efficiency of bare
liposomes and NKsomes were analyzed by measuring the DOX absorbance
at 490 nm. For cumulative drug release study, DOX-loaded NKsomes
(DOX@NKsome) and bare liposome were investigated in physiological
(PBS, pH 7.4) and acidic (Acetate buffer, pH 5.5) condition at
37.degree. C. In brief, 25 .mu.g/mL of samples were placed in 12-14
kDa dialysis membrane bag and dialyzed in the 250 mL of the
corresponding buffer at constant stirring (100 rpm). After regular
intervals of time, 500 .mu.L of samples were taken from the setup
and added a fresh buffer of the same volume to the setup. The
amount of DOX in the samples was quantified spectrofluorimetrically
by measuring DOX excitation and emission of 490 nm and 580 nm.
1.8. In Vitro Targeting Assay
[0111] The targeting ability of NKsomes against human breast cancer
cell MCF-7 and the normal human osteoblast cell NHost were tested
using flow passage assay. In brief, 1.times.10.sup.6 cells were
seeded onto the cell culture treated flow cells (ibid .mu.-slides)
and grown for 24 h at 37.degree. C. To mimic blood flow condition,
cells were then treated with Rh-labelled NKsomes and bare liposome
(at the concentration of 1.times.10.sup.12 nanoparticles suspended
in the overall volume of 3 mL of respective media) and were
passaged at a constant flow rate of 0.2 dyn/cm.sup.2 for different
flow cycles (6 cycles in 2 h). The accumulation of Rhodamine
fluorescence in the cells were recorded periodically by imaging the
cells in a different area under a fluorescent microscope equipped
with live cell imaging. After six cycles of a flow condition, the
cells were washed, fixed and immunostained with anti-CD56 for the
presence of NK-membrane and observed under CLSM.
1.9. In Vitro Immunogenicity Assay
[0112] The immunogenicity of NKsomes in THP-1 cells were tested for
its inflammatory response using a cytokine release assay. In brief,
7.5.times.10.sup.5 THP-1 cells were seeded in 12-well plate and
treated with isolated NKM (100 .mu.g/mL), NKsomes (100 .mu.g/mL),
Free DOX (5 .mu.g/mL), DOX@NKsomes (equivalent DOX load) and Bare
liposomes (100 .mu.g/mL) for 24 h incubation. Positive (LPS, 3
.mu.g/mL) and negative controls were maintained for assessing
pro-inflammatory cytokines in THP-1 cells. The treated cell culture
supernatants were collected, centrifuged to remove cell debris,
aliquot and stored at -20.degree. C. until needed. Samples were
thawed and used for analyzing pro-inflammatory cytokines,
IL-1.beta. (0.8 pg/ml), IL-6 (0.4 pg/mL) and TNF-.alpha. (0.7
pg/mL) using quantitative enzyme-linked immunosorbent assay KIT
(ELISA) (R&D Systems, Inc. Minneapolis, Minn.) as recommended
by the manufacturer. Sample fluorescence was measured at 450 nm
with the wavelength correction at 540 nm using Synergy H1 hybrid
microplate reader (BioTek Instruments Inc. VT).
1.10. In Vitro Anti-Cancer Effects
[0113] The in vitro cytotoxicity of DOX@NKsome and the equivalent
Free DOX were investigated against MCF-7 cells and NHost using a
cell cycle analysis and an MTT assay. For cell cycle analysis,
1.times.10.sup.6 cells were treated with 5 .mu.M of Free DOX,
equivalent DOX@NKsomes and bare NKsomes (equivalent to DOX-loaded)
for 12 h. After incubation, cells were harvested, fixed in 70%
ethanol overnight, washed with PBS twice and then followed
propidium iodide staining. Further, the samples were analyzed in
FACS Calibur for cell cycle analysis.
[0114] For MTT analysis, MCF-7 cells and NHost cells were treated
with NKsomes with different DOX concentration along with free DOX
and bare NKsomes. In brief, 2.times.10.sup.4 cells per well in the
respective medium were seeded in a 96-well plate and incubated for
24 h. While the cell reaches 80% confluence, the media was replaced
with different DOX concentration of free DOX and NKsomes (0.5, 1.5,
3, 5, 10 and 15 uM) and incubated for additional 24 h. Control
cells were maintained without DOX treatment. After incubation, MTT
dye was added according to the manual instruction and further
incubated for 3 h. The insoluble formation crystals were
solubilized using DMSO, and the absorbance was read at 590 and 630
nm using microplate reader (BioTek, Synergy H1 Hybrid reader).
1.11. Circulation Half-Life and Bio-Distribution Study.
[0115] The circulation half-life profile of NKsomes was
investigated using Six-week-old immunodeficient female NU/NU nude
mice (n=3). In brief, 5 mg/kg DiR-labelled NKsomes were
administered intravenously via tail vein injection, and the blood
samples were collected at predetermined time intervals (0.5, 1, 2,
4, 8, 12, 24, and 48 h) through the tail puncture. The blood
samples were analyzed under spectrofluorometer for the
quantification of DiR signals with the excitation and emission
wavelength of 750 and 780 nm. For the bio-distribution study, 5
mg/kg of DiR-labeled NKsomes was administered intravenously into
the MCF-7 tumor-bearing mice through tail vein injection, and after
24 h, animals were sacrificed, and the blood, heart, lung, spleen,
liver, kidney and tumor were isolated. The collected organs were
weighed, homogenized and quantified for the presence of DiR dye
using spectrofluorometry with the excitation and emission
wavelength of 750 and 780 nm. The stability of DiR in the
formulation was investigated in vitro in physiological condition to
assure its intactness with nanoconstruct.
1.12. In Vivo Anti-Tumor Therapy
[0116] The anti-tumor efficacy of DOX@NKsomes was determined along
with free DOX and unloaded NKsomes (aka "empty" NKsomes) using
MCF-7 induced solid tumor model in immunodeficient NU/NU nude mice
(n=3). In brief, 1.times.10.sup.6 MCF-7 cells in PBS was injected
subcutaneously into the hind rear flank region of the mice and the
tumor growth is monitored periodically. When the tumor size reached
4-5 mm, animals received 4 cycles of drug treatment at Day 1, 4, 7
and 10 [DOX@NKsomes and free DOX (equivalent DOX concentration of 5
mg/kg)] up to 3 weeks. Control groups were also maintained without
any treatment and bare NKsomes (10 mg/kg). During the study, body
weight and tumor volume of the animal groups were monitored
periodically to assess the therapeutic effects. The tumor volume
(V) was determined using V=L.times.W2/2, where L=length of the
tumor and W=width of the tumor. At the end of the study, animals
were euthanized, collected tumors and measured its size and weight.
Further, the tumor tissues were embedded in OCT, cryosectioned and
subjected to immunofluorescence for the qualitative accumulation of
NKsomes in tumor tissues by assessing the NKM protein marker (CD
56).
1.13. Statistical Analysis
[0117] Depending on the parameters One-way and Two-way ANOVA were
used for the statistical analysis. All data represent
mean.+-.standard deviation. ***P<0.001, **P<0.01, *P<0.05
were considered statistically significant.
[0118] 2. Results and Discussion
2.1. Preparation and Characterization of Fusogenic NKsomes
[0119] Preparation of NKsome involves the extraction of natural
killer (NK) cell membrane with surface receptor proteins isolated
from the activated NK-92 cells and surface infusion with synthetic
liposome as illustrated in FIG. 1. The membrane extraction was
carried using sucrose density gradient ultracentrifugation of the
whole cell extract (FIG. 2A). The isolated NK membrane was found at
the 30-40% interface of the sucrose gradient. The purified NK cell
membrane (NKM) was quantified for its protein content through
Bradford Assay. The average protein content in NK cell membrane
extracted from 1.times.10.sup.7 cells was found to be .about.300 ug
of protein. Further, to confirm the presence of integrated
characteristic membrane proteins (CD56, NKG-2D, and NKp30) of NK-92
cells in isolated NKM, a Dot-blot assay was carried out. The result
showed that characteristics proteins were successfully retained in
the purified NKM (FIG. 2G). For NKsome preparation, fusogenic
liposome was prepared using DOTAP:DOPE:Cholesterol in the molar
ratio of 50:45:5 by film hydration technique following published
protocols. The prepared liposome was extruded with NKM at different
protein concentration in three formulations by varying the protein
and lipid ratio viz.; NKsome-A 1:100, NKsome-B 1:500, and NKsome-C
1:1000, through 200 nm pore sized polycarbonate membrane filter.
The assembly of NKM fuses with the cationic lipid components of the
liposome driven by electrostatic interactions and thus forms stable
NKsomes. To confirm the fusion of NKM with the liposomes, a FRET
study was conducted by preparing FRET liposomes with fluorescence
donor
(L-.alpha.-phosphatidylethanolamine-N-(4-nitro-benzo-2-oxa-1,3-diazole)
(egg-transphosphatidylated, chicken), (PE-NBD) and a fluorescent
acceptor (L-.alpha.-phosphatidylethanolamine-N-(lissamine rhodamine
B sulfonyl) (ammonium salt) (PE-RhB) as building blocks of
liposome. FRET study utilizes the energy transfer mechanism to
identify the molecular distance between the fluorophores. Energy
transfer can occur from donor to the acceptor when they are in
proximity, thus minimizes the donor energy. As expected, when NKM
infused with the FRET liposomes a FRET effect was diminished. The
fluorescent intensity of the donor was increased whereas that of
acceptor was decreased. This is only possible when NK cell membrane
fuses with the FRET liposomes and increase in the distance between
two fluorophores. Further, increase in the concentration of protein
induce rapid aggregation of the NKsomes, thereby affecting its
stability in biological media.
[0120] Transmission electron microscopic examination shows that the
bare liposome and NKsome are spherical with the average diameter of
80 and 70 nm, respectively (FIG. 2B). It is important to note that
TEM size is in dry state measurement, which possibly distracted the
actual size and shape. The hydrodynamic diameter is the actual
diameter of NKsome under hydrated condition that was used in all in
vitro and in vivo experiments, which was found to be different
under various protein content viz., 85.+-.1, 88.+-.1, and 98.+-.0.6
nm for NKsome-A, NKsome-B, and NKsome-C, respectively, whereas that
of the bare liposome (without NKM) was 121.+-.2 nm in diameter.
These results clearly evident that the size of the NKsome was
reduced with the increased protein concentration, suggested the
fusogenic flexibility of liposome surface and protein and
phospholipids form NKM stabilized NKsome in narrow size (FIG. 2C).
The zeta potential of the isolated NKM was found to be -22.+-.0.3
mV, whereas the zeta potential of the prepared NKsome was found to
be -1.3.+-.0.2, +14.+-.0.3, and +22.5.+-.1.3 mV for NKsome-A,
NKsome-B, and NKsome-C, respectively, and that of bare liposome was
found to be +35.4.+-.0.8 mV. As compared to the positive zeta
potential of the bare cationic liposome, the zeta potential of
NKsome shows a successive reduction in cationic nature, which
further confirms the successful inclusion of NKM in NKsomes (FIG.
2D). Further, the stability and functional integrity of the NKsome
was tested by thermogravimetric analysis (TGA) and Fourier
transform-infrared (FT-IR) spectroscopy by analyzing lyophilized
NKsome samples. The TGA weight loss results under nitrogen
environment show that the synthetic liposome completely decomposed
at 450.degree. C., as no residual matter left at the end of TGA
analysis. Whereas, in typical NKM a significant residual protein
(.about.35%) remained at the analyzed temperature range. In
contrast, NKsome shows 15% of residual protein at the analyzed
temperature range, which further confirms the inclusion of NKM into
the synthetic liposome (FIG. 2E). Moreover, FT-IR spectrum shows
the presence of signature amide I band and carbohydrate region of
NKM in the NKsome thereby confirming the successful translocation
of NKM into the NKsomes (FIG. 2F). Out of three formulations,
NKsome-B shows enthusiastic colloidal stability than the other
formulations. Therefore, we choose this formulation for further
characterization and functional studies.
2.2. Protein Characterization of NKsomes
[0121] The biomimetic properties of the NKsome rely on the
characteristic surface property of NK cell membrane, which is well
known for its selective tumor homing ability and its role in
immunosurveillance of cancer or stressed cells. This property will
purely depend on the surface protein (NKG-2D, NKp30, etc.)
expression on NK-cells. Although FT-IR analysis shows the presence
of functional groups of these proteins, we further confirmed the
presence of surface marker proteins using SDS-PAGE and western blot
analysis. The SDS-PAGE reveals the total protein profiles of whole
cell lysate and the isolated NKM. The protein profiles of the NKM
was mostly retained in the prepared NKsome (FIG. 3A). It is
well-known that the NK-92 can target cancer cells specifically
through its surface receptor proteins like NKG2-D, NKp30, etc., for
effective immunotherapy. Western blot was carried out to confirm
the presence of signature protein receptors of NK-92 cells in
NKsomes. As shown in FIG. 3B, NKG2-D, NKp30 were retained in the
NKsomes along with its characteristic proteins like CD56, showing
the successful translocation of signature proteins for specific
targeting ability (FIG. 3B) thereby supporting the evidence
obtained from FT-IR. Also, the representative transmembrane
glycoprotein pan-cadherin was detected from the NKsome and served
as the control (FIG. 3B).
2.3. Fusogenic Characteristics of NKsomes
[0122] Recent progress in the area of fusogenic liposomes using
neutral and positive charged lipids in combination with .pi.
electron system has greatly enhanced the transfection efficiency of
biomolecules and drugs. As an alternative of endocytosis mediated
cellular uptake, fusogenic liposomes are more advantageous in
delivering therapeutics to the target cells by escaping lysosomal
degradation process. To investigate the fusogenic property of
NKsomes, NKsome-B was chosen due to its moderate cationic surface
charge and robust stability in comparison to other formulations. A
FRET study was conducted by preparing FRET NKsomes using
PE-NBD/PE-RhB and were allowed to fuse with the MCF-7 cell at room
temperature and investigated its fusogenic property. After 5 min of
incubation, it was clearly evident that the fluorescent intensity
of the acceptor reduced with the increase in the fluorescent
intensity of donor (FIG. 3C). This confirms the fusogenic potential
of NKsomes with cell membranes and thus retains its fusogenic
potential even after NKM infusion. Further, the time-lapse fusion
study of FRET NKsomes with MCF-7 cells showed steady increases in
the fluorescent emission of donor NBD within 5 min of incubation
and remains constant F1/F0 (F0=initial fluorescence intensity;
F1=fluorescence intensity over time), confirmed the successful
fusion of NKsomes with the cell membrane of the MCF-7.
[0123] Further to understand the fusogenic property of NKsomes in
vitro, MCF-7 cells were incubated with Rhodamine-labelled NKsome
(RhB-NKsome) under cell culture condition for 3 h. After
incubation, cell samples were collected and processed in FACS for
the characteristic NK cell markers like NKG-2D, NKp30, and CD56. As
shown in FIG. 3D, characteristic surface marker proteins NKG-2D,
NKp30, and CD56 signals were identified in NKsome treated MCF-7
cells, which further confirmed the fusogenic potential of NKsome
with the MCF-7 cells. FIG. 3E represents the CLSM micrographs of
RhB-NKsome fused MCF-7 cells stained for anti-CD65-FITC. As shown
in FIG. 3E, Rh-labelled fusogenic liposome and CD-56 markers were
found intact in the NKsome fused MCF-7 cells; this further supports
our proposed mechanism for drug delivery (FIG. 1). Also, Z-stack
confocal images further confirmed that the NKsome was fused only on
the surface of the MCF-7 cells. Whereas cells treated with NKM
coated, conventional anionic liposome showed no signs of NKM
accumulation in MCF-7 (FIG. 3F). With all these evidence, it was
clear that the NKsomes have potential fusogenic property to proceed
further with therapeutics.
2.4. Stability, Drug Loading, and Release Characteristics
[0124] The colloidal stability of NKsome was investigated at the
physiological conditions. FIG. 4A shows the stability of NKsome-B
at 4.degree. C. The result showed the sustained hydrodynamic size
of NKsomes even after two weeks. Further, its serum stability was
also investigated using 90% FBS. FIG. 4B shows the serum stability
of NKsomes at 37.degree. C. for 3 h conducted following published
protocols. Serum stability measures the increase in optical density
(OD) of serum at 560 nm due to the rapid aggregation of particles
when bound to serum proteins, which also indicate the possible
protein corona formation onto the surface of the particle. The
result showed that there was no significant change in the OD at 560
nm when NKsome was treated with serum, whereas the bare cationic
liposome showed a higher level of aggregation as revealed by
increased OD at 560 nm. Similarly, the stability of purified NKM
showed no signs of aggregation under serum condition. The stability
of NKsomes-A and C showed a drastic change in its hydrodynamic size
due to its NKM load. Therefore, NKsomes-B formulation was optimized
and used for further characteristic studies. The drug loading and
release kinetics of NKsomes were compared with the bare liposome
using the chemotherapeutic drug, DOX. FIG. 4C shows the comparative
encapsulation efficiency of NKsome and bare liposome at different
drug input concentrations (25-200 .mu.g/mL). No significant change
in the DOX loading efficiency was observed between bare liposomes
and NKsomes at the tested concentration (25-200 .mu.g/mL). The
maximum DOX encapsulation efficiency of the NKsome was found to be
80.+-.5%. DOX-loaded NKsome (DOX@NKsomes) are highly stable in
physiological conditions up to two weeks. The DOX release profile
from the NKsomes and bare liposome were also investigated in PBS
buffer (pH 7) and acetate buffer (pH 5.5) at 37.degree. C. (FIG.
4D). NKsome showed nearly 50% drug release even after 72 h, whereas
bare liposome showed burst release of 80% at physiological pH 7.
Under acidic condition (pH 5.5), NKsome showed nearly 75% drug
release whereas bare liposomes show 88% drug release in 72 h. These
results confirm the sustained drug release property of Nksomes
under physiological pH and its differential release under acidic
environment depicting its rapid release in the acidic tumor
microenvironment.
2.5. In Vitro Targeting Efficacy of NKsomes
[0125] Since NK cells are well known for its tumor targeting
properties, the in vitro targeting efficacy of NKsomes were
investigated using flow passage assay. In this method, tumor cells
and normal cells in in vitro conditions were subjected to
RhB-NKsome under flow condition at 37.degree. C. for 2 h. The
differential targeting efficacy of the NKsome with the human normal
osteoblast cells (NHost) and the human breast cancer cell (MCF-7)
was observed under continuous flow condition. FIG. 5A demonstrates
the accumulation of RhB-NKsome in MCF-7 cells over various cycles
of flow, under tested conditions. Each cycle of flow takes 20 min
interval, and the accumulation of red signal in the monolayer of
the cells indicates the targeting efficacy of NKsome against MCF-7.
The prepared RhB-Nksomes shows concentration-dependent fluorescence
in aqueous condition (FIG. 5B). In the case of NHost, no
significant amount of NKsome was accumulated, indicating the
differential targeting efficiency of NKsome over normal and a tumor
cell. FIG. 5C shows the quantitative analysis of NKsome
accumulation in MCF-7 and NHost determined by measuring the
Rhodamine fluorescence using ImageJ software (NIH, USA). It was
clear evidence that the accumulation of NKsome was found to be more
in MCF-7 than that of NHost. With the control bare liposome, no
significant difference was observed between MCF-7 and NHost (FIG.
5E and FIG. 5F). This selectivity of NKsome is attributed due to
the presence of NK cells protein markers, which can recognize and
binds to the receptors present in MCF-7. Further, the cells were
subjected to immunofluorescence staining with anti-CD56/FITC at the
end of the flow passage assay and imaged under a confocal
microscope. As expected, NKsome was accumulated more in the MCF-7
as proven by the FITC signals of CD56 (FIG. 5D). In the case of the
bare liposome, no significant accumulation was observed between
MCF-7 and NHost under flow condition. These results confirmed the
in vitro breast cancer cell targeting efficacy of NKsome.
2.6. In Vitro Immunogenicity and Therapeutic Effects of
NKsomes.
[0126] The immunogenicity of the NKsomes was further investigated
in human peripheral blood monocyte cells, THP-1 using human
pro-inflammatory cytokines ELISA Kit (IL-10, IL-6, and
TNF-.alpha.). Pro-inflammatory cytokines are vital biomarkers of
immunogenicity, often screened to calculate the immunomodulatory
effects of nanoformulations. FIG. 6 shows the immunogenicity
assessment of NKsomes treated with THP-1 cells after 24 h
incubation. The results showed that no significant immunogenicity
of NKsomes was observed in THP-1 cells after 24 h incubation. In
comparison with DOX@NKsomes, Bare liposomes, and NKM, only free DOX
shows the little elevation of IL-10 and TNF-.alpha. levels in THP-1
cells, this is presumably due to the immunoregulatory potential of
chemotherapeutic drug DOX (FIG. 6).
[0127] The in vitro therapeutic efficacy of DOX@NKsome was compared
with the equivalent amount of free DOX in MCF-7 and NHost using MTT
assay and cell cycle analysis. FIGS. 7A and 7B shows the in vitro
cytotoxicity with varying the concentration of DOX in NKsome and
free DOX after 24 h incubation. From the results, the significant
difference in cell viability was observed between free DOX and
DOX@NKsomes against MCF-7 and NHost. Dose-dependent cytotoxicity
was observed in both cases, whereas superior toxicity of free DOX
was predominant in NHost. This is possibly due to the higher tumor
targeting affinity of NKsomes with MCF-7 than that of NHost.
Control cell treated with bare NKsomes shows higher
biocompatibility even at the higher concentration (FIG. 7C).
Further, the therapeutic response of DOX@NKsome in MCF-7 and NHost
were investigated using cell cycle analysis. It has been well known
that DOX cause cell cycle arrest in G0/G1 phase and induce
apoptosis in cancer cells. FIG. 7D represents the cell cycle
histograms of MCF-7 and NHost cells treated with Free DOX and
DOX@NKsomes (Dox concentration=5 .mu.M) for 12 h. As expected, Free
DOX and DOX@NKsomes showed increased accumulation of cells in G2
phase with a decrease in G1 phase. In MCF-7, free DOX and
DOX@NKsomes treated cells shows a decrease in the G1 fraction (53%
and 54%, respectively) as compared to that of untreated and bare
NKsome (61% and 61%, respectively) (FIG. 6 D). Also increased
accumulation in G2 fraction (25 and 26%) was observed as compared
to that of untreated and bare NKsome (20% and 20%) (FIG. 6 E). In
NHost, free DOX and DOX@NKsomes treated cells shows a G1 fraction
of 53.3% and 61%, whereas untreated and bare NKsome treated cells
shows 64.3% and 63.9% (FIG. 6D). Further, increased accumulation in
G2 fraction (23.6 and 20.1%) were observed, as compared to that of
untreated and bare NKsomes treated cells (18.5% and 19.4%) (FIGS.
7E and 7F). Overall, no significant changes were observed between
MCF-7 and NHost when treated with Free DOX for 12 h. In
DOX@NKsomes, significant difference was observed in G1 and G2 phase
of MCF-7 and NHost cells after 12 h incubation.
2. 7. Circulation Half-Life and Bio-Distribution of NKsomes.
[0128] To determine the circulation half-life and biodistribution,
NKsome was incorporated with DiR
(1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide)
dye as a part of the liposomal building block and investigated in
NU/NU mice. The formulated DiR-NKsomes were found to be more stable
in physiological conditions. FIG. 8A shows the circulation
half-life profile of NKsome. The concentration of NKsome in the
blood was expressed as a percentage of injected dose (% ID). Higher
levels of NKsome was observed in the bloodstream over 8 h
post-injection (.about.60%), which described the distribution phase
of the NKsomes. After 8 h, it reduced further with time and showed
a clear trend of elimination phase. By two-compartmental
pharmacokinetic model, the half-life of the NKsomes was found to be
18 h in NU/NU mice. FIG. 8A, the inset shows the semi-log plot of
NKsomes pharmacokinetics which can be used to derive the
circulation half-life of NKsomes from the slope of semi-log signals
using one-way nonlinear elimination model. By nonlinear elimination
model, the circulation half-life of the NKsomes was found to be 8
h. Irrespective of the pharmacokinetic models, the circulation
half-life of the NKsome had longer circulation half-life and
reduced in vivo clearance, compared to the bare liposome. It is
evident that the biological membrane coatings of the nanoparticle
have longer circulation half-life than the normal nanoparticles.
Further, the biodistribution and tumor homing efficiency of NKsome
was investigated in MCF-7 induced solid tumor in NU/NU mice. After
24 h of intravenous injection of DiR-labeled NKsome, major organs
were collected and quantified (FIG. 8B). The result showed that the
NKsome was distributed mainly to tumors and reticuloendothelial
system organs. It is evident from the previous in vivo reports that
cationic liposome of DOTAP/DOPE/DOX formulation fabricated with
aptamer tends to accumulate more in liver, spleen, and kidney.
About 5% of the total injected dose reached the tumor tissues,
showing the tumor homing efficacy of NKsomes in NU/NU mice. It is
attributed that the NK-92 cell tends to accumulate more in the
tumor microenvironment, which largely depends on the overexpression
of NK cell receptor ligands like NKG2-D ligands in tumor cells.
Recent studies on Macrophage membrane coated liposome also shows
superior targeting of metastatic lung cancer model as like of
macrophage. Further, the tumor tissues were investigated through
immunofluorescence for the qualitative presence of NK-92 cell
membrane (NKM) by staining with FITC conjugated anti-CD-56. FIG. 8C
shows the presence of NKM in MCF-7 induced solid tumor in NU/NU
mice. Green fluorescence signals of FITC showed the accumulation of
NKM in tumor tissues confirming the in vivo tumor homing potential
of NKsome in NU/NU mice.
2.8. In Vivo Tumor Homing and Therapeutic Efficacy
[0129] With the assurance of longer blood circulation half-life and
efficient tumor targeting ability of NKsome, we move forward to
conduct a pilot therapeutic study. The in vivo therapeutic efficacy
of DOX@NKsomes was investigated against human breast cancer MCF-7
induced solid tumor in NU/NU mice. FIG. 9 shows the antitumor
efficacy of DOX@NKsomes against solid tumor model in comparison
with Free DOX and bare NKsome. The body weight of the tumor-bearing
animals of all groups was not significantly changed during the
treatment period (FIG. 9B). Control groups were also maintained
without any treatment. However, tumor volume of free DOX and
DOX@NKsomes, have been changed significantly in comparison with the
bare NKsome and untreated control animals. Animals treated with
both Free DOX and DOX@NKsomes shows a significant reduction in
tumor volume at the end of treatment period, whereas bare NKsomes
shows tumor proliferation (FIG. 9A). The tumor inhibition rate (IR
%) of the bare NKsome, Free DOX, and DOX@NKsome treated groups were
found to be 24.29, 63.69 and 78.5%, respectively (Table 2)
TABLE-US-00003 TABLE 2 Calculated tumor inhibition rate among
different treatment groups. Treatment groups Inhibition Rate
(%).sup.# Bare NKsomes 24.29 Free DOX 63.69 DOX@NKsomes 78.50
.sup.#IR % = (Control tumor weight-Treatment tumor weight)/Control
tumor weight * 100
Compared to free DOX, DOX@NKsomes exhibits enhanced antitumor
activity, augmenting its tumor targeting potential for cancer
therapy (FIGS. 8C and D). The average tumor weight for the
untreated control was found to be 0.6.+-.0.19 g, whereas bare
NKsome, Free DOX, and DOX@NKsomes treated animals were found to be
0.5.+-.0.05, 0.3.+-.0.04, and 0.2.+-.0.07 g, respectively (FIG.
9C). Compared to control and bare NKsomes, tumor weight of free DOX
and DOX@NKsomes showed reduced tumor mass. Further, the tumor
tissues were sectioned, immunostained for the qualitative detection
of NKsomes and DOX using confocal microscopy. FIG. 9E shows the
representative immunofluorescence image of DOX@NKsomes treated
tumor tissue sections, illustrating the distribution of NKsome
(FITC-anti-CD 56) and DOX (red fluorescence). This further assured
the tumor homing ability of NKsome for potential anti-cancer
therapy. FIG. 9F shows the Representative dot-blot analysis of
tumor tissue of DOX@NKsome treated animals for the qualitative
determination of its characteristics proteins (CD 56, NKG2D, and
MIC-A/B).
[0130] 3. Conclusion
[0131] In summary, we fabricated fusogenic NKsome made up of
activated natural killer membrane fused with the cationic liposome
capable of targeting tumor cells more efficiently in vitro and in
vivo conditions. The tumor targeting efficacy of NKsome was solely
depended upon the membrane characteristics of NK-92 cell membrane
receptors. The fabricated NKsome was found to be non-immunogenic,
more stable under physiological conditions, and capable of loading
chemotherapeutic drug, DOX, for targeted cancer therapy. Also,
NKsome exhibit prolongs circulation half-life and tumor homing
potential as demonstrated by biodistribution and pharmacokinetic
studies. Further, the DOX@NKsomes showed excellent anti-tumor
potential against human breast cancer cells, MCF-7 in vitro and in
vivo. Overall, this study demonstrated the tumor homing potential
of NKsome for targeted tumor therapy by exploiting the properties
of the natural killer cell membrane, which could open a new door
for design consideration in biomimetic nanomedicine.
Example 2
Biomimetic Natural Killer Membrane Camouflaged Polymeric
Nanoparticle for Targeted Bioimaging
[0132] In the present study, we designed a tumor targeting
biomimetic nanoconstruct (BNc) made up of the NKM camouflaged onto
the surface of carboxylate terminated polylactic-co-glycolic acid
(PLGA) NP. To this BNc, phospholipid-conjugated GBCA and NIR dye
was incorporated and studied their feasibilities under MRI and
further supported by NIR fluorescent imaging. The NKM was isolated
from the NK-92 cells and hybridized with imaging components and
PLGA NP using membrane extrusion technique. This technique gives us
the opportunity to tune magnetic relaxivity by varying the
gadolinium-lipid concentration onto the BNc. Considering the
acquired properties of NK cell, we hypothesized that the engineered
BNc would have an ability to function as NK cell, which could help
in maximizing the delivery of payloads, herein contrast agents,
into the tumor by co-working with blood pool agents, hence
enhancing the diagnostic efficiency in targeted cancer
bioimaging.
Materials and Methods
Materials
[0133] Poly (D,L-lactide-co-glycolide) carboxylate end group (50:50
dL/g) was purchased from DURECT Corporation (USA).
L-.alpha.-Phosphatidylethanolamine-N-(lissamine rhodamine B
sulfonyl) (Ammonium Salt) and (Egg-Transphosphatidylated, Chicken)
(Egg Liss Rhod-PE) were purchased from Avanti Polar Lipid Inc.
(USA). Gd (III) acetate was purchased from Alfa Aesar (USA).
Primary antibodies for NKG2-D, NKp30 and Pan-cadherin were procured
from Santa Cruz Biotechnology. NCAM (CD56) and Secondary HRP-linked
anti-mouse IgG antibody were purchased from Cell Signaling.
Fluorophore-conjugated antibodies like FITC Mouse anti-Human CD56
and .kappa. Isotype Control were purchased from BD Bioscience. All
other reagents and chemicals used were of analytical grade.
Cell Lines and Tumor Models
[0134] Human NK cells, NK-92, were procured from ATCC, Manassas,
USA. NK-92 cells were grown and regularly passaged in Alpha Minimum
Essential Medium (.alpha.-MEM) without ribonucleosides and
deoxyribonucleosides and supplemented with 1.5 g/L sodium
bicarbonate, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, 0.02 mM
folic acid, 0.2 mM inositol, 200 U/ml recombinant IL-2, 12.5% (v/v)
horse serum and 12.5% (v/v) fetal bovine serum. The human breast
cancer cells, MCF-7, were procured from ATCC (USA). The cells were
grown and maintained in Dulbecco's Modified Eagle's Medium (DMEM)
supplemented with 10% (v/v) fetal bovine serum (FBS),
penicillin/streptomycin (100 ug/ml) and maintained at 37.degree. C.
in 5% CO.sub.2 environment.
[0135] For tumor studies, six-week-old female immunodeficient NU/NU
nude mice were procured from Charles River Laboratories
International, Inc. (USA). All animal experiments and protocols
were approved by the Institutional Animal Care and Use Committee
(IACUC) and Institutional Biosafety Committee (IBC), Kansas State
University, Manhattan. For solid tumor models, 1.times.10.sup.6
MCF-7 cells in saline were implanted subcutaneously into the hind
rear flank region of the mice and the tumor growth were monitored
periodically.
Isolation of NK Cell Membrane
[0136] Isolation of NKM were performed using sucrose gradient
ultracentrifugation method as reported. In brief, nearly
.about.2.times.10.sup.8 NK cells, NK-92, grown in multiple T-75
culture flask were harvested and washed twice with 1.times.PBS.
Further, the washed cell pellet was suspended in homogenization
buffer (10 mM Tris-HCl, 1 mM MgCl.sub.2, 1 mM KCL, 2 mM PMSF, 25 mM
Sucrose, 200 .mu.g/mL Trypsin-chymotrypsin Inhibitor, 10 .mu.g/mL
DNase and 10 .mu.g/mL RNase) and homogenized in ice for 5 min (20 s
pulse and 30 s in between pulses). The homogenized suspension was
pooled over the discontinuous sucrose gradient (55%, 40% and 30%
(w/v) sucrose in 0.85% saline) in polycarbonate tubes and
ultracentrifuged in a Beckman SW 27 rotor at 28,000 g for 30 min at
4.degree. C. The NKM fraction at 30% to 40% interface was collected
in a clean tube and analyzed for its protein characterization using
the dot-blot technique. For purification, collected membrane
fractions were diluted with twice the volume of normal saline and
ultra-centrifuged in a Beckman SW 27 rotor at 28,000 g for 1 h at
4.degree. C. The purified membranes were lyophilized, weighed,
quantified for its protein content using Bradford Assay and stored
at 4.degree. C. for further use.
Preparation of Biomimetic Nanoconstruct
[0137] BNc were prepared using a nanoprecipitation method. In
brief, 1 mg of PLGA dispersed in acetonitrile was added drop-wise
to different concentrations of Gd-lipid (50-400 ug) dispersed in 2
ml of 4% ethanol under magnetic stirring at 60.degree. C. After 15
min, 1 ml of Milli-Q water was added to cool down the suspension
and further stirred at room temperature. After 1 h of stirring, the
clear nanoparticle suspension (PLGA core) was extruded in the
presence of isolated NKM (100 ug dispersed in PBS) using 100 nm
pore-size Millipore membrane filter, concentrated in Amicon
centrifugal filter (10 kDa), and stored at 4.degree. C. for further
use. For DiR/Rhodamine labeling, 10 .mu.g of the dye dispersed in
chloroform was dissolved in the PLGA dispersion and followed the
same protocol of the preparation of BNc.
Characterization of BNc
[0138] The hydrodynamic size and zeta potential of the prepared BNc
were characterized using Dynamic light scattering analysis
(Malvern, Nano ZSP). The size and the shape of the prepared BNc
were confirmed using transmission electron microscope (FEI Technai
G2 Spirit BioTWIN). Further, the concentration of Gd.sup.3+ in BNc
were determined using an inductively coupled plasma mass
spectrometry (ICP-MS, NEXion 350X, Perkin Elmer). The concentration
of proteins in all samples was determined using Bradford Assay
following manufacture's recommendation.
Protein Characterization of BNc
[0139] For SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel
Electrophoresis), all samples were prepared with the overall
protein concentration of 50 .mu.g/wells loaded in a 4-20%
Mini-PROTEAN.RTM. TGX Protein Gels and stained with Coomassie
brilliant blue. For western blot, protein samples were transferred
to the PVDF membrane and treated with primary antibodies for CD-56
(Cell Signaling), NKG2-D (Santa Cruz), NKp30 (Santa Cruz) and
Pan-cadherin (Santa Cruz) along with HRP-conjugated anti-mouse IgG
secondary antibody (Cell Signaling). The blotted films were further
developed using SignalFire.TM. ECL Reagent (Cell signaling) and
imaged chemiluminescent signals Bio-imager (Kodak).
Stability of BNc
[0140] The physiological stability of the prepared BNc at 4.degree.
C. in PBS (pH=7) was investigated using dynamic light scattering
size analysis. In brief, 50 .mu.g/mL of BNc samples in PBS were
incubated at 4.degree. C. for two weeks and investigated their
change in size by measuring the samples in DLS every day. The serum
stability of the prepared BNc was carried out as reported earlier.
In brief, 100 .mu.L of 500 .mu.g/mL of BNc were mixed with 100
.mu.L of 90% FBS at 37.degree. C. and record their change in
absorbance with incubation time kinetically by recording at every 3
sec over a period of 3 h using Microplate reader (BioTek, Synergy
H1 Hybrid reader).
Gd.sup.3+ Release Study
[0141] The cumulative Gd.sup.3+ release characteristics of BNc
under the physiological condition at 37.degree. C. was determined
periodically. In brief, 50 .mu.g/mL of BNc was placed in a 12-14
Kda dialysis membrane bag and dialyzed against 250 mL of PBS
(pH=7). At constant agitation (70 rpm), 200 .mu.L of the buffer
samples were collected at predetermined time intervals (0-72 h) and
replace with an equivalent volume of fresh PBS buffer. The amount
of Gd released from the BNc is determined using ICP-MS as reported
before.
In Vitro Magnetic Properties of BNc
[0142] The MRI relaxivity of Gd-loaded BNc was determined using
published protocols. The longitudinal relaxation (LR) time of BNc
in the presence and the absence of NKM in an equivalent Gd.sup.3+
concentration (10 .mu.g/mL) was determined using a RARE (Rapid
Acquisition with Relaxation Enhancement) pulse sequence with
variable repetition time on a 14.1 T NMR system (Bruker Avance III,
600 MHz NMR-MRI). Concentration-dependent recovery curve of BNc was
also investigated using a different concentration of BNc (0.05-0.4
mM). The LR was determined from the T.sub.1 relaxation time and the
concentration of Gd.sup.3+. The corresponding T.sub.1 weighted
magnetic resonance phantom images were also recorded using a turbo
spin echo sequence (TR=1500 ms, TE=6.50 ms, and slice thickness=1
mm).
Cellular Uptake and Biocompatibility Studies
[0143] The cellular uptake efficiency of BNc in the presence and
the absence of the NKM coating were investigated using FACS
analysis (BD FACSCalibur.TM.). In brief, 3.times.10.sup.6MCF-7
cells were grown in a T25-flask and treated with 50 .mu.g/mL of
Rhodamine-labelled BNc dispersed in DMEM media. After 6 h of
incubation, cells were trypsinized, washed and analyzed in FACS for
the quantitative determination of NP uptake by MCF-7 cells. For the
confocal study, Rh-labelled BNc were treated in an 8-chambered
micro-chamber slide seeded with the cell density of 50,000
cells/well and incubated for 3 h. After incubation, cells were
washed, fixed, and immunostained with FITC-anti-CD 56. Nuclei of
the cells were stained with DAPI, and the slides were observed
directly in a Confocal Laser Scanning Microscope (Carl Zeiss,
LSM-700). Further, the biocompatible nature of the BNc was
investigated in MCF-7 cells using the
[3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide]
(MTT) Assay. In brief, MCF-7 cells were seeded in a 96-well plate
at the density of 10,000 cells/well and incubated for 24 h. After
confluence, cells were treated with different concentration of BNc
(10-150 .mu.g/mL) and incubated for additional 24 h. After
incubation, the medium was removed, 10 .mu.L of MTT (5 mg/mL) dye
were added and incubated further for 3 h in the dark at 37.degree.
C. After 3 h incubation, the formazan crystals were dissolved using
DMSO, and the plates were read for absorbance at 590 nm using
Microplate reader (BioTek, Synergy H1 Hybrid reader).
In Vitro Cytokine Release Assay
[0144] The immunogenicity of the prepared BNc along with the bare
PLGA NP were tested for its immunoregulatory potential using
standard cytokine release assay. In brief, 8.times.10.sup.5 THP-1
cells were seeded in a 12-well plate and treated with bare PLGA NP
(100 .mu.g/mL), NKM (100 .mu.g/mL), Gd-lipid (100 .mu.g/mL) and BNc
(100 .mu.g/mL) at 37.degree. C. for 24 h. After 24 h incubation,
cell culture supernatants were collected, centrifuged, removed cell
debris and stored at -20.degree. C. as small aliquots. For cytokine
assay, samples were thawed and analyzed for pro-inflammatory
cytokines, IL-1.beta. (LOD: 1.7 pg/ml), IL-6 (LOD: 1.5 pg/mL), and
TNF-.alpha. (LOD: 1 pg/mL) using Magnetic Human Cytokine Multiplex
Assays Kit (R&D Systems, Inc. Minneapolis, Minn.) in Luminex
MagPix.RTM. instrument (Millipore Inc.,) as per the manufacturer
recommendations. For positive control, cells were dosed with 3
.mu.g/mL of lipopolysaccharide (LPS) for 24 h.
Pharmacokinetics and Bio-Distribution of BNc
[0145] The circulation half-life and pharmacokinetic profile of the
BNc were investigated using Six-week-old female NU/NU nude mice
(n=3). In brief, BNc (5 mg/kg) were administered intravenously via
tail vein injection, and the blood samples were collected at
predetermined time intervals (0.5-48 h) through tail vein puncture.
The amount of BNc in the blood samples was quantified using ICP-MS
as described earlier. For the bio-distribution study, 5 mg/kg of
BNc were injected i.v. into the MCF-7 tumor-bearing mice, and after
24 h of study, animals were sacrificed to collect major organs and
tumor tissues. The tissue samples were weighed, digested using 2 mL
of aqua regia (3:1 ratio of HCl:HNO.sub.3), diluted in 2% HNO.sub.3
and analyzed for the Gd concentration in ICP-MS as described
earlier. Pharmacokinetic parameters were analyzed by a
two-compartmental model using the MATLAB software (MathWorks,
2017b).
Mathematical Model
[0146] Pharmacokinetic models are employed to illustrate the
process of nanodrug distribution in the whole body. When we follow
a particular nanodrug administered with a specific route, we may
study the whole body as a kinetically single unit, conventionally
termed as compartment, which is homogeneous. For this purpose, we
assume that the administered drug distributes uniformly in the body
and that the drug equilibrates between different tissue/organ in a
time-dependent fashion. However, we cannot conclude that the
concentration of drug is the same in tissue or plasma. The
bicompartmental model divides body into two different units or
compartments. This division helps us study each tissue and its
interaction with its relative components. In bicompartmental model,
we assume that the administered drug enters the first compartment
and then transported into the second compartment, tissue or
organ..sup.[39] The remaining drug will come back to the
compartment 1 and then we have elimination of drug from first
compartment.
[0147] In this study, we assumed that disposition of drug from
blood to tissue follows a bicompartmental model. For the
bicompartmental model, we assume that we have a first order
transfer rate between two compartments, k.sub.23 and k.sub.32, and
we consider a first order elimination rate from the second
compartment, k.sub.el, without any elimination or metabolism in the
tissue.
In Vivo Bio-Imaging
[0148] For in vivo bio-imaging studies, near-infrared fluorescent
dye 1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindotricarbocyanine
Iodide (DiR) labeled BNc (10 mg/kg) were injected intravenously
into the MCF-7 tumor-bearing mice along with bare control PLGA NP
and analyzed its bioaccumulation and tumor targeting efficiency
using Pearl.RTM. Trilogy Small animal imaging system (LI-COR.RTM.).
The fluorescent images at the near-infra-red window were taken at
pre-determined time intervals (0, 3, 6, 12 and 24 h) and the images
were analyzed in Image Studio.RTM. software. At the end point, mice
were euthanized, and excised organs were imaged and analyzed for
the quantitative determination of BNc accumulation.
Ex Vivo MRI Imaging
[0149] A pilot ex vivo MRI study was performed using NU/NU mice
bearing MCF-7 tumors on a 14.1 T NMR system (Bruker Avance III, 600
MHz NMR-MRI, 14.1 T). In brief, NU/NU mice were intravenously
injected with BNc (equivalent Gd concentration of 0.008 mmol/kg),
and the animals were sacrificed after 2 h post-injection. Within 15
min of the sacrifice time, animals were imaged in MRI. The
T.sub.1-weighted MR images were recorded using a QTR 30 mm coil
with a FLASH (Fast, slow angle shot) protocol at 37.degree. C. The
axial MR imaging parameters were TR=1500 ms, TE=6.5 ms, flip
angle=90, image size 256.times.256, FOV=30.times.30 and slice
thickness=0.5 mm. Further, the ex vivo MR images were analyzed
using DICOM software (Santesoft Ltd). All animal experiments and
protocols were approved by the Institutional Animal Care and Use
Committee and Institutional Biosafety Committee, Kansas State
University, Manhattan.
Statistical Analysis
[0150] All experimental data represent the mean.+-.standard
deviation. Depending on the experiment parameters One-way and
Two-way ANOVA were used for the statistical analysis.
***P<0.001, **P<0.01, *P<0.05 were considered
statistically significant.
Results and Discussion
Preparation and Characterization of BNc
[0151] BNcs were fabricated using membrane extrusion method in the
manner similar to that of liposomal fabrication. The process
involves the preparation of PLGA NP, extraction and isolation of
NKM, and the cloaking of PLGA NP with isolated NKM, GBCA, and NIR
dye. The biocompatible polymeric NP, PLGA, was prepared using
nano-precipitation following our earlier reports. Further, the NKM
was isolated and extracted from the NK-92 cells through sucrose
gradient centrifugation. The extracted NKM was lyophilized and
re-dispersed in PBS. The amount of NKM yield was quantified by
measuring the protein concentration. From .about.2.times.10.sup.8
NK-92 cells, the NKM yield was found to be .about.300 .mu.g protein
equivalent. Finally, the BNc were fabricated by extruding PLGA NP
and Gd-lipid with isolated NKM [with the weight ratio of (5:1:1)]
through 200 nm pore sized polycarbonate membrane. For NIR dye
labeling, constant 1.0 wt % of dye with respect to the 1 mg of PLGA
was used in all cases. Gd.sup.3+ conjugated phospholipid (Gd-lipid)
was synthesized via simple convenient coupling chemistry as
described in earlier reports. FIG. 10A illustrates the schematic
representation of the process of BNc fabrication. The hydrodynamic
size of the bare PLGA NP was found to be 109.+-.2.8 nm, whereas the
BNc were found to be in the range of 110.+-.20 nm, respectively
(FIG. 10B). The transmission electron microscopic images of the BNc
show clear coating materials onto the surface of the PLGA NP (FIG.
10C). Further, to investigate its fabrication efficiency, three
different formulations of BNc were fabricated as represented in
Table 3 as follows: BNc-A=(1000 .mu.g PLGA):(200 .mu.g
Gd-lipid):(20 .mu.g NKM); BNc-B=(1000 .mu.g PLGA):(200 ug
Gd-lipid):(100 ug NKM); BNc C=(1000 ug PLGA):(200 ug Gd-lipid):(200
ug NKM).
TABLE-US-00004 TABLE 3 Physicochemical characteristics of BNc.
Hydrodynamic Zeta Nanoparticles Size (d nm) PDI Potential (mV) BNc
A (5:1:0.1) * 111 .+-. 2.1 0.16 .+-. 0.02 -20.7 .+-. 0.76 BNc B
(5:1:0.5) * 128 .+-. 1.6 0.19 .+-. 0.01 -36.9 .+-. 4.07 BNc C
(5:1:1) * 134 .+-. 4.4 0.28 .+-. 0.04 -41.1 .+-. 0.59 Bare PLGA NPs
(5:1) * 109 .+-. 2.8 0.14 .+-. .0.01 -20.1 .+-. 0.82 NKM NA NA
.sup. -26 .+-. 0.21 * Ratios indicate in BNc corresponding to
PLGA:GD-lipid:NKM protein ratio (by weight) NA sample not
measured.
The results from hydrodynamic size analysis indicated that the size
of the BNc slightly increases with the increase in the
concentration of NKM, which is presumably due to the presence of a
larger amount of NKM that becomes hydrated in an aqueous
environment. Further, the zeta potentials of each construct
demonstrate that the coating of NKM changes its zeta potential
value more negative among different formulations, which is
attributed to the negative zeta potential of the cell membrane that
was organized onto the negatively charged PLGA NP. Based on
physicochemical characteristics, more stable BNc-C (5:1:1) were
chosen for further characterization studies. Hereafter, BNc
represents BNc-C. Next, we identified signature proteins in the BNc
using SDS-PAGE and western blot analysis. FIGS. 11A and 11B
represent the SDS-PAGE and western blot of the isolated NKM along
with the engineered BNc. SDS-PAGE analysis revealed that the
proteins from the NKM were successfully retained in the BNc. Among
the proteins, the characteristic signature proteins such as CD56,
NKG-2D, and NKp30 were identified from the western blot analysis
along with the control pan-cadherin (FIG. 11B). NKG-2D and NKp30
are activating receptors found on the NK-cells for effective
cytolytic functions on the tumor cell, which overexpress their
ligands. NK cells recognize tumor cells and stressed cells through
these ligands. Thus, the presence of these signature proteins would
facilitate the tumor targeting, thereby delivering maximum contrast
agents to help distinguish tissue contrast.
[0152] The stability of BNc in in vivo serum/plasma conditions was
investigated by storing the BNc in PBS at 4.degree. C. and
measuring the variation in hydrodynamic size for 14 days. FIG. 12A
shows the stability of BNc after 14 days in PBS. No significant
change in its size was observed over 14 days of study. Further, the
serum stability of the BNc was investigated by incubating the BNc
with 90% FBS, FIG. 12B. Compared to bare PLGA NP, BNc show higher
stability in serum conditions as evident from the no change in its
optical density over the period at 560 nm. This optical measurement
records the change in absorbance due to particle aggregation.
[0153] The Gd.sup.3+ loading in BNc was further investigated in
detail by increasing the concentration of Gd-lipid input during BNc
fabrication. With the fixed concentration of PLGA and NKM (5:1),
the concentration of Gd-lipid was varied between 50 and 400 ug/mg
of PLGA (FIG. 12C). The maximum Gd loading efficiency of BNc was
found to be .about.19%, and the Gd loading content was found to be
4.5.+-.0.1 .mu.g/mg of PLGA. However, we observed that the higher
loading of Gd.sup.3+ results in instability of BNc in physiological
pH. For further studies, BNc with the Gd.sup.3+ content of 3.+-.0.4
.mu.g with the loading efficiency of 11% were used for all other
experiments (BNc). Further, the Gd.sup.3+ release characteristics
of BNc were investigated at pH=7.4 in PBS as well as at pH=5 in
acetate buffer in order to map the Gd.sup.3+ stability in BNc which
is directly related to toxicity. We further assumed that by
stabilizing Gd.sup.3+ in BNc, Gd.sup.3+ associated toxicity would
be reduced as its ionic form is toxic. A cumulative Gd.sup.3+
release from the BNc was performed by dialyzing the samples using
500 Da dialysis bag, which gives easy passage to release free
Gd.sup.3+ (Mw 157.25 Da) to the dialysis reservoir. The cumulative
release percentage of Gd.sup.3+ from BNc at pH=5, 7.4, and 5.2 was
found to be around 2% and 10% after 48 h (FIG. 12D). This further
confirms the stability of Gd.sup.3+ in the fabricated BNc.
[0154] FIG. 13 shows the magnetic resonance properties of BNc
dispersed in water. The r.sub.1 relaxivity of the BNc in the
presence and the absence of NKM was found to be 4.8.+-.0.6
mM.sup.-1s.sup.-1 and 5.0.+-.0.5 mM.sup.-1s.sup.-1, respectively
(FIG. 13 A). We did not observe significant differences in the
r.sub.1 relaxivity, which further assures us that the distribution
of Gd-lipid in the BNc is not affected by the NKM coating. This is
in agreement with the earlier report where authors discussed
alternation in r.sub.1 relaxivity due to the distance induced
confinement of Gd.sup.3+. In the present case, if NMK has inserted
in between Gd-lipids instead of surface coating, this would
increase the distance between Gd-lipids, and we should experience a
significant change in relaxivity. FIG. 13B shows the MR phantom
images of different concentrations of BNc acquired at 14.1 T. The
phantom images show brighter contrast as the concentration
increases to 0.4 mM, this was further confirmed by the
corresponding T.sub.1 curve (FIG. 13C).
[0155] The cellular uptake and intracellular distribution of the
Rhodamine labeled BNc were investigated in MCF-7 cells using
confocal laser scanning microscope and flow cytometry (FIG. 14).
MCF-7 cells treated with BNc show rapid uptake of the BNc within 3
h of incubation. The uptaken BNc were found intact in the cells as
evident from the bioaccumulation of immunostained FITC-anti-CD56
and Rhodamine signals inside the cells (FIG. 15). The nuclei of the
cells were further stained with DAPI, and this shows BNc were
distributed in perinuclear regions. Z-stack images of MCF-7 show
clear evidence of intracellular distribution of BNc (FIG. 14A).
Further, the selective targeting of the BNc in MCF-7 cells was
compared with the bare PLGA NP labeled with Rhodamine dye. Flow
cytometry analysis was conducted to assess the cellular uptake
efficiency. The results indicate that cellular uptake of BNc was
more predominant than the bare PLGA NP illustrating the targeting
potential of BNc towards cancer cells (FIG. 14B). The
biocompatibility of the BNc and bare PLGA NP was investigated in
MCF-7 cells through MTT assay. 24 h incubation results show that
even at higher concentration (150 .mu.g/mL) BNc were not cytotoxic
(FIG. 14C). This clearly demonstrated the biocompatible nature of
BNc, which can be used for bioimaging applications in vivo.
[0156] Further, the immunogenicity of the NKM derived BNc was
investigated in human peripheral blood monocytes, THP-1, using
pro-inflammatory cytokine release assay (FIG. 16). Pro-inflammatory
cytokines (IL-10, IL-6, and TNF-.alpha.) are important biomarkers
to identify the immunoregulatory potential of nanoformulations. As
documented in FIG. 16, engineered BNcs are non-immunogenic as they
exhibit minor response against IL-1.beta., IL-6, and TNF-.alpha. in
comparison with the control cells and positive control (FIG. 16).
No significant levels of immunogenicity were observed for isolated
NKM, bare PLGA NP, Gd-lipid in comparisons with the control
cytokine levels (FIG. 16). Only the positive control, LPS (3
.mu.g/mL), shows the elevated levels of IL-1.beta., IL-6, and
TNF-.alpha. in the tested cells. This clearly confirms the
immuno-compatibility of BNc for further in vivo imaging
studies.
[0157] With the assurance of BNc colloidal stability, in vitro
cancer targeting, and biocompatibility, we moved forward to
understand their in vivo targeting ability in immunodeficient NU/NU
nude mice bearing an MCF-7 tumor. As shown in FIG. 17A, DiR labeled
BNc (10 mg/kg) were injected via tail vein, and the distribution of
particles was studied under live animal imaging over a period of 24
h. Distinguishable kinetics of BNc accumulation in the tumor from
3, 6, 12, and 24 h, as shown by the increase in fluorescent
intensity in the tumor, further suggests the tumor targeting
ability of BNc. Accumulation of BNc over the 24 h period also
suggests us the blood availability of BNc, thereby providing us a
key to its long circulating properties. After 24 h of the imaging
session, mice were euthanized, and the major organs were collected.
These collected organs were washed with PBS and subjected to ex
vivo fluorescent imaging (FIG. 17B). E vivo imaging proves the
overall tissue distribution of BNc with major accumulation in tumor
and RES organs like liver and spleen. Compared to the control group
(bare PLGA NP), BNc show significant accumulation in the tumor
which is twice as much as that of bare PLGA NP (FIG. 17C).
[0158] Next, in vivo pharmacokinetics of BNc was investigated after
a single intravenous injection of 5 mg/kg of NP in MCF-7
tumor-bearing immunodeficient NU/NU nude mice. We used Gd.sup.3+as
a handle using ICP-MS to assess the pharmacokinetics and additional
biodistribution studies. For this purpose, BNc were concentrated
using Amicon Ultra centrifuge unit (3000 Da molecular weight
cutoff) and characterized for its hydrodynamic size and weight
yield after lyophilization. Both size and weight of BNc were found
to be consistent in a number of experiments conducted blindly by a
research technician. FIG. 18A shows the blood clearance of BNc
after 72 h. The blood retention of BNc was found be high (60%) in
the first 10 h and it gradually reduced with time. The amount of
BNc in the blood was determined using ICP-MS analysis. The
biodistribution efficiency of BNc was investigated after a single
intravenous administration of BNc (5 mg/kg). After 24 h, animals
were euthanized, and the major organs were investigated for the
presence of BNc by estimating organ content of Gd using ICP-MS. The
percentage of BNc in the liver and the spleen was found to be high,
as they are the principal reticuloendothelial system that clears NP
from the body. The percentage of BNc in the tumor tissue was found
to be 10.+-.3% of the injected dose (FIG. 18B). This further
confirms the tumor affinity of NKM fabricated BNc for targeted
bioimaging applications and supports our results for NIR imaging
session. The pharmacokinetic parameters of the BNc were obtained by
fitting the concentration of BNc in blood and tissues with respect
to time intervals and dosage using a two-compartmental model
(MATLAB 2017b). Table 4 shows the pharmacokinetic parameters of BNc
in MCF-7 tumor-bearing NU/NU mice.
TABLE-US-00005 TABLE 4 Pharmacokinetic parameters of BNc injected
NU/NU mice by two-compartmental analysis. PK parameters Biomimetic
Nanoconstruct (BNc) t.sub.1/2 (h) 9.51 .+-. 5.74 AUC (% ID h/mL)
1068.8 .+-. 507.27 Vd (mL) 3.85 .+-. 1.16 CL (mL/h) 0.28 .+-. 0.14
MRT (h) 13.6 .+-. 1.38 t.sub.1/2: half-life, AUC: Area under curve,
Vd: Volume of distribution, CL: Clearance, MRT: Mean residence
time
[0159] Finally, the advantage of NKM coated BNc in MR imaging of
tumors were demonstrated using 14.1 T NMR system (Bruker Avance
III, 600 MHz NMR-MRI). For ex vivo MR imaging, following our
approved IACUC protocol, tumor animals were intravenously injected
with BNc (equivalent Gd concentration of 0.008 mmol/kg) and
euthanized after 2 h. Animals were euthanized and then, following
our approved protocol for MRI, rapidly imaged for T.sub.1-weighted
MR image contrast, recorded using a QTR 30 mm coil with a FLASH
protocol at 37.degree. C. FIG. 18C shows the T.sub.1-weighted MR
images slice of NU/NU mice treated with BNc (negative control MRI
in FIG. 19). Compared with different image contrast of Gd
standards, illustrated in different color circles (red: water,
yellow: 18 .mu.M Magnevist.RTM., blue: 37 .mu.M Magnevist.RTM.),
BNc shows distinctive brighter image contrast in tumor tissues.
Along the various MRI slices (slice thickness of 500 .mu.m), it is
clearly evident that the BNc were deeply penetrated into the tumor
tissues and shown enhanced MR image contrasts to evaluate tumor as
compared to that of surrounding soft tissue contrast. Comparing the
data from MRI ex vivo imaging and data from in vivo NIR bioimaging,
the engineered BNc show promises in targeting MCF-7 tumor.
Conclusion
[0160] Herein, we described a biomimetic approach to fabricate a
multifunctional NP system that has acquired properties from NK
cells and studied its potential for tumor targeting and imaging.
This hybrid platform technology, where synthetic and biological
components were merged to exhibit unique properties, and consisting
of both hydrophilic and hydrophobic assembly, shows versatile
properties and feasibilities in surface functionalization and have
a vacancy in the hydrophobic core for cargo encapsulation.
Moreover, the existence of BNc in the cancer cells and the tumor as
compared to that of bare particle suggested the dependence of
targeting due to the acquired properties from the NK-92 cells.
Considering the fact that successful cancer therapy requires drugs
being precisely delivered to the tumors, the engineered BNc would
have promises in drug delivery and monitor the therapeutic response
in a single session.
Example 3
Method of the Double Emulsion to Load Water-Soluble Drug
[0161] Water soluble drugs can be encapsulated into NK-PLGA by a
water-in-oil-in-water double-emulsion solvent evaporation
technique. First, an aqueous solution containing water-soluble drug
was emulsified with a solution of the polymer in chloroform. The
resulting water-in-oil first emulsion is the droplet of drug
enclosed in the polymer mixture. Immediately, the first emulsion
was further emulsified with an aqueous solution of the Natural
killer cell membrane to form an oil-in-water second emulsion. Under
these water-in-oil-in-water circumstances, NPs that formed were
stabilized by NK membrane phospholipid and protein. The interior of
the particle contains the water-soluble drug. The synthesis
protocol was optimized to obtain homogeneous and well dispersed
spherical NPs. In a typical experiment, the first emulsion
(water-in-oil) was prepared by probe sonication of a mixture of 1
mg of water-soluble drug in 1 mL water with 10 mg PLGA in mL of
chloroform. Immediately after the formation of the first emulsion,
it was further emulsified with a mixture containing 2 mg NK
membrane protein in 10 mL of water (oil-in-water) under probe
sonication for 5 min resulting in the formation of
water-in-oil-in-water double emulsion. This mixture was kept under
stirring overnight to evaporate chloroform from the emulsion. After
complete evaporation of chloroform, NPs formed were washed with
deionized water using Amicon ultra centrifugal filter (Mw cut
off=10 kDa) to remove un-encapsulated drug.
Example 4
Method to Coat Natural Killer Membrane onto Metallic
Nanoparticle
[0162] The NK-92 membrane was collected using aforementioned
protocol. The NK-92 membrane coated metallic nanoparticles can be
achieved by bath sonication or by extrusion as discussed above.
Examples include but are not limited to 15 nm gold nanoparticles
(AuNPs).
Method
[0163] Synthesis of 15 nm AuNPs: 15 nm AuNPs were synthesized by a
modified Turkevich method. Briefly, 25 mL of 1 mM of gold(III)
chloride hydrate (HAuCl.sub.4) solution was heated to reflux at
190.degree. C. To the boiled HAuCl.sub.4 solution, 2.5 of 40 mM
sodium citrate tribasic hydrate solution was added. The mixture was
allowed to stir and reflux for 30 minutes. The solution should
slowly turn to ruby red colored indicating the formation of 15 nm
AuNPs. After 30 minutes, the heating was stopped and mixture was
left to stir for 2 hrs. The size of AuNPs can be tuned from 5 nm to
100 nm by varying the types and amount of reducing agents (sodium
citrate, sodium borohydride) and amount of gold seeds. FIG. 20A
shows the prepared citrate gold nanoparticles.
[0164] AuNPs, larger in size and/or shape than 15 nm can be
prepared by using 15 nm AuNPs as a seed using seed mediated growth
mechanism.
[0165] NK-92 membrane coated AuNPs: NK-92 membrane coated AuNPs can
be achieved using bath sonication technique. In brief, as prepared
AuNPs solution was centrifuged at 3000 g for 15 minutes to
concentrate to 6 mL and removed excess amount of sodium citrate.
Thereafter, 1 mL of purified AuNPs was sonicated with 60 .mu.g
NK-92 membrane for 10 minutes at 50% power. The result NK-AuNPs was
centrifuge at 5000 rpm for 5 minutes to remove uncoated membrane.
FIG. 20B shows TEM images of the NK-AuNPs.
[0166] Characterization: The NK-AuNPs was subjected to dynamic
light scattering, zeta potential, stability, and TEM studies to
understand the physiochemical properties. FIG. 21 shows a graph of
the spectrophotometric characterization of the metal nanoparticles
before and after NKM coating. FIG. 22 shows a graph of the zeta
potential measurement of the coated and uncoated metal
nanoparticles. FIG. 23 shows a graph comparing the dynamic light
scattering analysis of coated and uncoated metal nanoparticles.
FIGS. 24A and 24B shows the TEM analysis of the NK-AuNPs.
[0167] Cellular interaction: The interaction between uncoated and
NK-coated AuNPs with breast cancer cell (MCF-7) was investigated
using TEM. In brief, cells were seeded in a T75 and maintained
under cell culture condition. When cells reach to 80% confluence,
AuNPs or NK-AuNP were added into cell media to get final
concentration of 30 .mu.g/mL. After 24 h of incubation, cells were
washed 3 times with PBS, trypsinized, and fixed in Trump fixative
reagent.
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